The present invention relates to cytotoxic agents bearing reactive polyethylene glycol linkers and methods for making such agents. These agents may be used in the production of cytotoxic conjugates, or for other purposes, such as in an affinity resin for use in the isolation of cellular components that recognize and bind the cytotoxic agents.
The present invention also relates to novel cytotoxic conjugates comprising polyethylene glycol linkers, methods of making the conjugates, and their therapeutic use. More specifically, the invention relates to novel cytotoxic conjugates comprising cytotoxic agents joined to cell-binding agents using hetero-bifunctional polyethylene glycol linkers, methods for making the conjugates and their therapeutic use. These novel cytotoxic conjugates have therapeutic use in that the cytotoxic portion of the conjugates can be delivered to specific cell populations in a targeted fashion, due to the linkage of the cytotoxic agent to a cell-binding agent.
Conjugates of highly cytotoxic agents, such as maytansinoids and CC-1065 analogs, and cell-binding agents have been shown to possess exceptional target-specific anti-tumor activity (U.S. Pat. Nos. 5,208,020, 5,416,064, 5,475,092, 5,585,499, and 5,846,545). In such cytotoxic conjugates, the cytotoxic agent is bound to the cell-binding agent via a disulfide bond or via a short disulfide-containing linker. It has been previously shown that such disulfide-containing linkers are both stable upon storage, and efficiently cleaved inside a tumor cell to release fully active drug (Liu et al., Proc. Natl. Acad. Sci. 93:8618-8623 (1996); Chari et al., Cancer Res. 55:4079-4084 (1995); Chari, R. V. J., Adv. Drug Delivery Rev. 31:89-104 (1998)). Cleavage likely occurs via disulfide exchange between the disulfide-linked cytotoxic agent and an intracellular thiol, such as glutathione.
However, because most highly-potent cytotoxic agents used in cytotoxic conjugates are hydrophobic, two technical difficulties arise. The first is that conjugation reactions between cytotoxic agents and cell-binding agents require reaction conditions that address the hydrophobic nature of the cytotoxic agents. These conditions include very dilute solutions, the use of large volumes, and the presence of large amounts of non-aqueous co-solvents, which may damage the proteinacious cell-binding agents. As a result, preparation and purification processes become quite cumbersome, and the final cytotoxic conjugate is obtained in low concentration, necessitating the administration of large volumes to patients.
The second technical difficulty is that cytotoxic conjugates prepared using disulfide bonds or short disulfide-containing linkers are only sparingly soluble in pharmaceutical solutions typically used for parenteral administration to patients. It is therefore difficult to produce formulations for such conjugates.
Thus, in order to develop improved methods for producing cytotoxic conjugates, and increase the flexibility in formulating pharmaceutical solutions containing the conjugates, there is a need to address the hydrophobicity of both the cytotoxic agents and the cytotoxic conjugates produced using cytotoxic agents.
One manner in which to meet both of these goals would be to develop novel linker molecules that allow the hydrophobic cytotoxic agents to be manipulated under aqueous conditions and permit the formation of a cytotoxic conjugate that is soluble under both aqueous and non-aqueous conditions.
Polyethylene glycol (PEG) has been found to be useful in the conversion of cytotoxic drugs into prodrugs, thereby extending the half-life of the drugs in circulation in vivo, and improving their water solubility (for a review see Greenwald, R. B., J. Controlled Release 74:159-171 (2001)). For example, the anti-cancer drug Taxol has been converted into the prodrug PEG-Taxol by linking PEG via an ester bond to the C-2xe2x80x2 position of Taxol (U.S. Pat. No. 5,824,701; Greenwald et al., J. Med. Chem. 39:424-431 (1996); Greenwald et al., J. Org. Chem. 60:331-336 (1995)).
However, PEG used in such applications is usually very large (average molecular weight of 40,000). Such size is required to significantly alter the pharmacokinetics of the drug. The drug molecules are also typically reacted with PEG via an ester or carbamate group of the drug, which can result in a drastic decrease in drug potency. In addition, the PEG moiety must be cleaved in vivo by some enzymatic mechanism to restore the activity of the drug, a process which is often inefficient. Finally, PEG used in such applications is typically mono-functional, i.e., only one terminus of the PEG molecule is modified so that it can be linked to the drug.
The present inventors have prepared novel PEG linking groups that provide a solution to both of the difficulties discussed above. These novel PEG linking groups are soluble both in water and in non-aqueous solvents, and can be used to join one or more cytotoxic agents to a cell-binding agent. The PEG linking groups are hetero-bifunctional linkers in that they bind to cytotoxic agents and cell-binding agents at opposite ends of the linkers through a functional sulfhydryl or disulfide group at one end, and an active ester at the other end. The linking groups have the two-fold advantage over other linking groups in that (1) they can be chemically joined to a cytotoxic agent in a non-aqueous solvent via a disulfide bond, thereby surmounting the hydrophobic nature of the agent and making it soluble in both non-aqueous and aqueous solvents, and (2) cytotoxic conjugates produced using the linking groups have greater solubility in water, thereby permitting much greater flexibility in the formulation of pharmaceutical solutions for parenteral administration to patients.
Thus, herein disclosed are cytotoxic agents bearing a PEG linking group having a terminal active ester, and cytotoxic conjugates comprising one or more cytotoxic agents joined to a cell-binding agent via a PEG linking group. In addition, a therapeutic composition comprising a cytotoxic conjugate is disclosed.
Also disclosed are methods of preparing cytotoxic agents bearing a PEG linking group having a terminal active ester, and methods of preparing cytotoxic conjugates comprising one or more cytotoxic agents joined to a cell-binding agent via a PEG linking group. Finally, a method for killing selected cell populations using cytotoxic conjugates is disclosed.
In one embodiment of the invention, cytotoxic agents bearing a polyethylene glycol (PEG) linking group having a terminal active ester are disclosed. The cytotoxic agents contemplated in this, and each proceeding embodiment, include a thiol-containing maytansinoid, thiol-containing taxane, thiol-containing CC-1065 analogue, thiol-containing daunorubicin analogue and thiol-containing doxorubicin analogue, and thiol-containing analogues or derivatives thereof. The core of the terminal active esters of the PEG linking group contemplated in this, and each proceeding embodiment, are esters that readily react with amino groups, including N-succinimidyl, N-sulfosuccinimidyl, N-phthalimidyl, N-sulfophthalimidyl, 2-nitrophenyl, 4-nitrophenyl, 2,4-dinitrophenyl, 3-sulfonyl-4-nitrophenyl or 3-carboxy-4-nitrophenyl esters.
In a preferred embodiment, the PEG linking group has from 1 to 20 monomeric units. In an equally preferred embodiment, the PEG linking group has from 21 to 40 monomeric units. In a further equally preferred embodiment, the PEG linking group has from 41 to 1000 monomeric units.
Specifically contemplated is a cytotoxic agent, bearing a polyethylene glycol (PEG) linking group having a terminal active ester and 1 to 20 monomeric units, of formula 2: 
wherein Z is said cytotoxic agent;
wherein Q is R2COOxe2x80x94, R2R3NCOOxe2x80x94, R2OCOOxe2x80x94, R2Oxe2x80x94, R2CONR3xe2x80x94, R2R3Nxe2x80x94, R2OCONR3xe2x80x94, or Sxe2x80x94,
xe2x80x83wherein:
R2 is SCR4R5R6xe2x80x94,
R4, R5 and R6 are each H, linear alkyl, cyclic alkyl or branched alkyl, and may be the same or different,
R3 is H or a linear alkyl, cyclic alkyl or branched alkyl;
wherein n is an integer of from 0 to 20;
wherein x is 1 or 2; and
wherein Y is N-succinimidyl, N-sulfosuccinimidyl, N-phthalimidyl, N-sulfophthalimidyl, 2-nitrophenyl, 4-nitrophenyl, 2,4-dinitrophenyl, 3-sulfonyl-4-nitrophenyl or 3-carboxy-4-nitrophenyl.
Also contemplated is a cytotoxic agent, bearing a PEG linking group having a terminal active ester and 21 to 40 monomeric units, of formula 2: 
wherein Z is said cytotoxic agent;
wherein Q is R2COOxe2x80x94, R2R3NCOOxe2x80x94, R2OCOOxe2x80x94, R2Oxe2x80x94, R2CONR3xe2x80x94, R2R3Nxe2x80x94, R2OCONR3xe2x80x94, or Sxe2x80x94,
xe2x80x83wherein:
R2 is SCR4R5R6xe2x80x94,
R4, R5 and R6 are each H, linear alkyl, cyclic alkyl or branched alkyl, and may be the same or different,
R3 is H or a linear alkyl, cyclic alkyl or branched alkyl;
wherein n is an integer of from 21 to 40;
wherein x is 1 or 2; and
wherein Y is N-succinimidyl, N-sulfosuccinimidyl, N-phthalimidyl, N-sulfophthalimidyl, 2-nitrophenyl, 4-nitrophenyl, 2,4-dinitrophenyl, 3-sulfonyl-4-nitrophenyl or 3-carboxy-4-nitrophenyl.
Also contemplated is a cytotoxic agent, bearing a PEG linking group having a terminal active ester and 41 to 1000 monomeric units, of formula 2: 
wherein Z is said cytotoxic agent;
wherein Q is R2COOxe2x80x94, R2R3NCOOxe2x80x94, R2OCOOxe2x80x94, R2Oxe2x80x94, R2CONR3xe2x80x94, R2R3Nxe2x80x94, R2OCONR3xe2x80x94, or Sxe2x80x94,
xe2x80x83wherein:
R2 is SCR4R5R6xe2x80x94,
R4, R5 and R6 are each H, linear alkyl, cyclic alkyl or branched alkyl, and may be the same or different,
R3 is H or a linear alkyl, cyclic alkyl or branched alkyl;
wherein n is an integer of from 41 to 1000;
wherein x is 1 or 2; and
wherein Y is N-succinimidyl, N-sulfosuccinimidyl, N-phthalimidyl, N-sulfophthalimidyl, 2-nitrophenyl, 4-nitrophenyl, 2,4-dinitrophenyl, 3-sulfonyl-4-nitrophenyl or 3-carboxy-4-nitrophenyl.
Also contemplated is the cytotoxic agent according to any one of the examples above, wherein said cytotoxic agent is selected from the group consisting of a thiol-containing maytansinoid, thiol-containing taxane, thiol-containing CC-1065 analogue, thiol-containing daunorubicin analogue and thiol-containing doxorubicin analogue, and thiol-containing analogues or derivatives thereof.
Also contemplated is the cytotoxic agent according to the relevant examples above, wherein said thiol-containing maytansinoid is a C-3 thiol-containing maytansinoid.
Also contemplated is the cytotoxic agent according to the relevant examples above, wherein said C-3 thiol-containing maytansinoid is an N-methyl-alanine-containing C-3 thiol-containing maytansinoid.
Also contemplated is the cytotoxic agent according to the relevant examples above, wherein said N-methyl-alanine-containing C-3 thiol-containing maytansinoid is a compound selected from the following formula M1: 
wherein:
l is an integer of from 1 to 10; and
may is a maytansinoid.
Also contemplated is the cytotoxic agent according to the relevant examples above, wherein said N-methyl-alanine-containing C-3 thiol-containing maytansinoid is a compound selected from the following formula M2: 
wherein:
R1 and R2 are H, CH3 or CH2CH3, and may be the same or different;
m is 0, 1, 2 or 3; and
may is a maytansinoid.
Also contemplated is the cytotoxic agent according to the relevant examples above, wherein said N-methyl-alanine-containing C-3 thiol-containing maytansinoid is a compound selected from the following formula M3: 
wherein:
n is an integer of from 3 to 8; and
may is a maytansinoid.
Also contemplated is the cytotoxic agent according to the relevant examples above, wherein said N-methyl-alanine-containing C-3 thiol-containing maytansinoid is a N-methyl-alanine-containing C-3 thiol-containing maytansinol.
Also contemplated is the cytotoxic agent according to the relevant examples above, wherein said N-methyl-alanine-containing C-3 thiol-containing maytansinol is a dechloro maytansinol.
Also contemplated is the cytotoxic agent according to the relevant examples above, wherein said N-methyl-alanine-containing C-3 thiol-containing maytansinol is a compound selected from the following formula M6: 
wherein:
l is 1, 2 or 3;
Y0 is Cl or H; and
X3 is H or CH3.
Also contemplated is the cytotoxic agent according to the relevant examples above, wherein said C-3 thiol-containing maytansinoid is an N-methyl-cysteine-containing C-3 thiol-containing maytansinoid.
Also contemplated is the cytotoxic agent according to the relevant examples above, wherein said N-methyl-cysteine-containing C-3 thiol-containing maytansinoid is a compound selected from the following formula M4: 
wherein:
o is 1, 2 or 3;
p is an integer of 0 to 10; and
may is a maytansinoid.
Also contemplated is the cytotoxic agent according to the relevant examples above, wherein said N-methyl-cysteine-containing C-3 thiol-containing maytansinoid is a N-methyl-cysteine-containing C-3 thiol-containing maytansinol.
Also contemplated is the cytotoxic agent according to the relevant examples above, wherein said N-methyl-cysteine-containing C-3 thiol-containing maytansinol is a dechloro maytansinol.
Also contemplated is the cytotoxic agent according to the relevant examples above, wherein said N-methyl-cysteine-containing C-3 thiol-containing maytansinol is a compound selected from the following formula M5: 
wherein:
o is 1, 2 or 3;
q is an integer of from 0 to 10;
Y0 is Cl or H; and
X3 is H or CH3.
Also contemplated is the cytotoxic agent according to the relevant examples above, wherein said thiol-containing taxane is a compound selected from the following formula T1: 
wherein:
R1 is H, an electron withdrawing group, or an electron donating group, and R1xe2x80x2 and R1xe2x80x3 are the same or different and are H, an electron withdrawing group, or an electron donating group;
R2 is heterocyclic, a linear, branched, or cyclic ester or ether having from 1 to 10 carbon atoms or a carbamate of the formula xe2x80x94CONR10R11, wherein R10 and R11 are the same or different and are H, linear, branched or cyclic alkyl having 1 to 10 carbon atoms or aryl;
R3 is an aryl, or a linear, branched, or cyclic alkyl having from 1 to 10 carbon atoms;
R4 is xe2x80x94OC(CH3)3 or phenyl;
R5 is a thiol moiety; and
R6 is heterocyclic, H, a linear, branched, or cyclic ester or ether having from 1 to 10 carbon atoms or a carbamate of the formula xe2x80x94CONR10R11, wherein R10 and R11 are the same or different and are H, linear, branched or cyclic alkyl having 1 to 10 carbon atoms or aryl.
Also contemplated is the compound of the relevant examples above, wherein R1 is F, NO2, CN, Cl, CHF2, CF3, xe2x80x94OCH3, OCH2CH3, or NR7R8, wherein:
R7 and R8 are the same or different and are linear, branched, or cyclic alkyl having 1 to 10 carbon atoms or simple or substituted aryl having 1 to 10 carbon atoms.
Also contemplated is the compound of the relevant examples above, wherein R7 and R8 each has 1 to 4 carbon atoms.
Also contemplated is the compound of the relevant examples above, wherein R7 and R8 are the same.
Also contemplated is the compound of the relevant examples above, wherein R7 and R8 are the same.
Also contemplated is the compound of the relevant examples above, wherein R2 is xe2x80x94COC2H5, xe2x80x94CH2CH3, xe2x80x94CONHCH2CH3xe2x80x94CO-morpholino, xe2x80x94CO-piperidino, xe2x80x94CO-piperazino, or xe2x80x94CO-N-methylpiperazino.
Also contemplated is the compound of the relevant examples above, wherein R5 is xe2x80x94(CH2)nS, xe2x80x94CO(CH2)nS, xe2x80x94(CH2)nCH(CH3)S, xe2x80x94CO(CH2)nCH(CH3)S, xe2x80x94(CH2)nC(CH3)2S, xe2x80x94CO(CH2)nC(CH3)2S, xe2x80x94CONR12(CH2)nS, xe2x80x94CONR12(CH2)nCH(CH3)S, xe2x80x94CONR12(CH2)nC(CH3)2S, xe2x80x94CO-morpholino-XS, xe2x80x94CO-piperidino-XS, xe2x80x94CO-piperazino-XS, or xe2x80x94CO-N-methylpiperazino-XS;
wherein n is an integer of 1 to 10; and
wherein R12 is H, a linear alkyl, branched alkyl or cyclic alkyl having 1 to 10 carbon atoms, or simple or substituted aryl having from 1 to 10 carbon atoms or heterocyclic.
Also contemplated is the compound of the relevant examples above, wherein R1 is in the meta position when R1xe2x80x2 and R1xe2x80x3 are H or OCH3.
Also contemplated is the compound of the relevant examples above, wherein R3 is xe2x80x94CHxe2x95x90C(CH3)2.
Also contemplated is the cytotoxic agent according to the relevant examples above, wherein said thiol-containing taxane is a compound selected from the following formula T1: 
wherein:
R1 is H, an electron withdrawing group, or an electron donating group, and R1xe2x80x2 and R1xe2x80x3 are the same or different and are H, an electron withdrawing group, or an electron donating group;
R2 is a thiol moiety;
R3 is an aryl, or is a linear, branched, or cyclic alkyl having from 1 to 10 carbon atoms;
R4 is xe2x80x94OC(CH3)3 or phenyl;
R5 is heterocyclic, H, a linear, branched, or cyclic ester or ether having from 1 to 10 carbon atoms or a carbamate of the formula xe2x80x94CONR10R11, wherein R10 and R11 are the same or different and are H, linear, branched or cyclic alkyl having 1 to 10 carbon atoms or aryl; and
R6 is heterocyclic, H, a linear, branched, or cyclic ester or ether having from 1 to 10 carbon atoms or a carbamate of the formula xe2x80x94CONR10R11 wherein R10 and R11 are the same or different and are H, linear, branched or cyclic alkyl having 1 to 10 carbon atoms or aryl.
Also contemplated is the compound of the relevant examples above, wherein at least one of R1 is F, NO2, CN, Cl, CHF2, CF3, xe2x80x94OCH3, xe2x80x94OCH2CH3, or NR7R8 wherein:
R7 and R8 are the same or different and are linear, branched, or cyclic alkyl having 1 to 10 carbon atoms or simple or substituted aryl having 1 to 10 carbon atoms.
Also contemplated is the compound of the relevant examples above, wherein R7 and R8 each has 1 to 4 carbon atoms.
Also contemplated is the compound of the relevant examples above, wherein R7 and R8 are the same.
Also contemplated is the compound of the relevant examples above, wherein R7 and R8 are the same.
Also contemplated is the compound of the relevant examples above, wherein R5 is xe2x80x94(CH2)nS, xe2x80x94CO(CH2)nS, xe2x80x94(CH2)nCH(CH3)S, xe2x80x94CO(CH2)nCH(CH3)S, xe2x80x94(CH2)nC(CH3)2S, xe2x80x94CO(CH2)nC(CH3)2S, xe2x80x94CONR12(CH2)nS, xe2x80x94CONR12(CH2)nCH(CH3)S, xe2x80x94CONR12(CH2)nC(CH3)2S, xe2x80x94CO-morpholino-XS, xe2x80x94CO-piperidino-XS, xe2x80x94CO-piperazino-XS, or xe2x80x94CO-N-methylpiperazino-XS;
wherein n is an integer of 1 to 10; and
wherein R12 is H, a linear alkyl, branched alkyl or cyclic alkyl having 1 to 10 carbon atoms, or simple or substituted aryl having from 1 to 10 carbon atoms or heterocyclic.
Also contemplated is the compound of the relevant examples above, wherein R5 is xe2x80x94COC2H5, xe2x80x94CH2CH3, xe2x80x94CONHCH2CH3, xe2x80x94CO-morpholino, xe2x80x94CO-piperidino, xe2x80x94CO-piperazino, or xe2x80x94CO-N-methylpiperazino.
Also contemplated is the compound of the relevant examples above, wherein R1 is in the meta position when R1xe2x80x2 and R1xe2x80x3 are H or OCH3.
Also contemplated is the cytotoxic agent according to the relevant examples above, wherein said thiol-containing taxane is a compound selected from the following formula T1: 
wherein:
R1 is H, an electron withdrawing group, or an electron donating group, and R1xe2x80x2 and R1xe2x80x3 are the same or different and are H, an electron withdrawing group, or an electron donating group;
R2 is heterocyclic, a linear, branched, or cyclic ester or ether having from 1 to 10 carbon atoms or a carbamate of the formula xe2x80x94CONR10R11, wherein R10 and R11 are the same or different and are H, linear, branched or cyclic alkyl having 1 to 10 carbon atoms or aryl;
R3 is an aryl, or is a linear, branched or cyclic alkyl having from 1 to 10 carbon atoms;
R4 is xe2x80x94OC(CH3)3 or phenyl;
R5 is heterocyclic, H, a linear, branched, or cyclic ester or ether having from 1 to 10 carbon atoms or a carbamate of the formula xe2x80x94CONR10R11, wherein R10 and R11 are the same or different and are H, linear, branched or cyclic alkyl having 1 to 10 carbon atoms or aryl;
R6 is a thiol moiety.
Also contemplated is the compound of the relevant examples above, wherein R1 is F, NO2, CN, Cl, CHF2, CF3, xe2x80x94OCH3, xe2x80x94OCH2CH3, or NR7R8 wherein:
R7 and R8 are the same or different and are linear, branched, or cyclic alkyl having 1 to 10 carbon atoms or simple or substituted aryl having 1 to 10 carbon atoms.
Also contemplated is the compound of the relevant examples above, wherein R7 and R8 each has 1 to 4 carbon atoms.
Also contemplated is the compound of the relevant examples above, wherein R7 and R8 are the same.
Also contemplated is the compound of the relevant examples above, wherein R7 and R8 are the same.
Also contemplated is the compound of the relevant examples above, wherein R2 is xe2x80x94COC2H5, xe2x80x94CH2CH3, xe2x80x94CONHCH2CH3, xe2x80x94CO-morpholino, xe2x80x94CO-piperidino, xe2x80x94CO-piperazino, or xe2x80x94CO-N-methylpiperazino.
Also contemplated is the compound of the relevant examples above, wherein R5 is xe2x80x94(CH2)nS, xe2x80x94CO(CH2)nS, xe2x80x94(CH2)nCH(CH3)S, xe2x80x94CO(CH2)nCH(CH3)S, xe2x80x94(CH2)nC(CH3)2S, xe2x80x94CO(CH2)nC(CH3)2S, xe2x80x94CONR12(CH2)nS, xe2x80x94CONR12(CH2)nCH(CH3)S, xe2x80x94CONR12(CH2)nC(CH3)2S, xe2x80x94CO-morpholino-XS, xe2x80x94CO-piperidino-XS, xe2x80x94CO-piperazino-XS, or xe2x80x94CO-N-methylpiperazino-XS;
wherein n is an integer of 1 to 10; and
wherein R12 is H, a linear alkyl, branched alkyl or cyclic alkyl having 1 to 10 carbon atoms, or simple or substituted aryl having from 1 to 10 carbon atoms or heterocyclic.
Also contemplated is the compound of the relevant examples above, wherein R1 is in the meta position when R1xe2x80x2 and R1xe2x80x3 are H or OCH3.
Also contemplated is the cytotoxic agent according to the relevant examples above, wherein said thiol-containing CC-1065 analogue is a cyclopropylbenzindole-containing cytotoxic compound formed from an A subunit of the formulae A-3 or A-4 covalently linked to either a B subunit of the formula F-1 or a B-C subunit of the formulae F-3 or F-7 via an amide bond from the secondary amino group of the pyrrole moiety of the A subunit to the C-2 carboxyl group of the B subunit,
wherein the formulae A-3 and A-4 are as follows: 
wherein the formulae F-1, F-3 and F-7 are as follows: 
wherein each Z may be the same or different and may be O or NH; and
wherein, in Formula F-1 R4 is a thiol moiety, in Formula F-3 one of R or R4 is a thiol moiety, in Formula F-7 one of Rxe2x80x2 or R4 is a thiol moiety; when R or Rxe2x80x2 is a thiol moiety, then R1 to R6, which may be the same or different, are hydrogen, C1-C3 linear alkyl, methoxy, hydroxyl, primary amino, secondary amino, tertiary amino, or amido; and when R4 is a thiol moiety, R, R1, R2, R3, R4, R5 and R6, which may be the same or different, are hydrogen, C1-C3 linear alkyl, methoxy, hydroxyl, primary amino, secondary amino, tertiary amino, or amido, and Rxe2x80x2 is NH2, alkyl, O-alkyl, primary amino, secondary amino, tertiary amino, or amido.
Also contemplated is the cytotoxic agent of the relevant examples above, wherein R and Rxe2x80x2 are thiol moieties and R1 to R6 are each hydrogen.
Also contemplated is the cytotoxic agent of the relevant examples above, wherein R or R4 is NHCO(CH2)lS, NHCOC6H4(CH2)lS, or O(CH2)lS, and Rxe2x80x2 is (CH2)lS, NH(CH2)lS or O(CH2)lS wherein:
l is an integer of 1 to 10.
Also contemplated is the cytotoxic agent of the relevant examples above, wherein R or R4 is NHCO(CH2)nS, NHCOC6H4(CH2)lS, or O(CH2)lS, and Rxe2x80x2 is (CH2)lS, NH(CH2)lS or O(CH2)lS wherein:
l is an integer of 1 to 10.
Also contemplated is the cytotoxic agent of the relevant examples above, wherein l is 1.
Also contemplated is the cytotoxic agent of the relevant examples above, wherein l is 2.
Also contemplated is the cytotoxic agent according to the relevant examples above, wherein said thiol-containing doxorubicin analogue is a compound selected from the following formula D2: 
wherein,
Y is O or NR2, wherein R2 is linear or branched alkyl having 1 to 5 carbon atoms;
R is a thiol moiety, H, or liner or branched alkyl having 1 to 5 carbon atoms; and
Rxe2x80x2 is a thiol moiety, H, or xe2x80x94OR1, wherein R1 is linear or branched alkyl having 1 to 5 carbon atoms;
provided that R and Rxe2x80x2 are not thiol moieties at the same time.
Also contemplated is the compound of the relevant examples above, wherein NR2 is NCH3.
Also contemplated is the compound of the relevant examples above, wherein Rxe2x80x2 is xe2x80x94O.
Also contemplated is the compound of the relevant examples above, wherein the thiol moiety is xe2x80x94(CH2)nS, xe2x80x94O(CH2)nS, xe2x80x94(CH2)nCH(CH3)S, xe2x80x94O(CH2)nCH(CH3)S, xe2x80x94(CH2)nC(CH3)2S, or xe2x80x94O(CH2)nC(CH3)2S, wherein n is an integer of 1 to 10.
Also contemplated is the cytotoxic agent according to the relevant examples above, wherein said thiol-containing daunorubicin analogue is a compound selected from the following formula D3: 
wherein,
Y is O or NR2, wherein R2 is linear or branched alkyl having 1 to 5 carbon atoms;
R is a thiol moiety, H, or liner or branched alkyl having 1 to 5 carbon atoms; and
Rxe2x80x2 is a thiol moiety, H, or xe2x80x94OR1, wherein R1 is linear or branched alkyl having 1 to 5 carbon atoms;
provided that R and Rxe2x80x2 are not thiol moieties at the same time.
Also contemplated is the compound of the relevant examples above, wherein NR2 is NCH3.
Also contemplated is the compound of the relevant examples above, wherein Rxe2x80x2 is xe2x80x94O.
Also contemplated is the compound of the relevant examples above, wherein the thiol moiety is xe2x80x94(CH2)nS, xe2x80x94O(CH2)nS, xe2x80x94(CH2)nCH(CH3)S, xe2x80x94O(CH2)nCH(CH3)S, xe2x80x94(CH2)nC(CH3)2S, or xe2x80x94O(CH2)nC(CH3)2S, wherein n is an integer of 1 to 10.
In a second embodiment of the invention, a cytotoxic conjugate comprising one or more cytotoxic agents linked to a cell-binding agent through a PEG linking group is disclosed. The cell-binding agents contemplated in this, and each proceeding embodiment, include antibodies (especially monoclonal antibodies and antibody fragments), interferons, lymphokines, hormones, growth factors, vitamins, and nutrient-transport molecules (such as transferrin).
In a preferred embodiment, the PEG linking group has from 1 to 20 monomeric units. In an equally preferred embodiment, the PEG linking group has from 21 to 40 monomeric units. In a further equally preferred embodiment, the PEG linking group has from 41 to 1000 monomeric units.
Specifically contemplated is a cytotoxic conjugate, comprising one or more cytotoxic agents covalently bonded to a cell-binding agent through a PEG linking group having 1 to 20 monomeric units, wherein a linkage of one of said one or more cytotoxic agents is illustrated in formula 3: 
wherein Z is said cytotoxic agent;
wherein Q is R2COOxe2x80x94, R2R3NCOOxe2x80x94, R2OCOOxe2x80x94, R2Oxe2x80x94, R2CONR3xe2x80x94, R2R3Nxe2x80x94, R2OCONR3xe2x80x94, or Sxe2x80x94,
xe2x80x83wherein:
R2 is SCR4R5R6xe2x80x94,
R4, R5 and R6 are each H, linear alkyl, cyclic alkyl or branched alkyl, and may be the same or different,
R3 is H or a linear alkyl, cyclic alkyl or branched alkyl,
wherein n is an integer of from 0 to 20;
wherein x is 1 or 2; and
wherein A is said cell-binding agent.
Also contemplated is a cytotoxic conjugate, comprising one or more cytotoxic agents covalently bonded to a cell-binding agent through a PEG linking group having 21 to 40 monomeric units, wherein a linkage of one of said one or more cytotoxic agents is illustrated in a formula 3: 
wherein Z is said cytotoxic agent;
wherein Q is R2COOxe2x80x94, R2R3NCOOxe2x80x94, R2OCOOxe2x80x94, R2Oxe2x80x94, R2CONR3xe2x80x94, R2R3Nxe2x80x94, R2OCONR3xe2x80x94, or Sxe2x80x94,
xe2x80x83wherein:
R2 is SCR4R5R6xe2x80x94,
R4, R5 and R6 are each H, linear alkyl, cyclic alkyl or branched alkyl, and may be the same or different,
R3 is H or a linear alkyl, cyclic alkyl or branched alkyl,
wherein n is an integer of from 21 to 40;
wherein x is 1 or 2; and
wherein A is said cell-binding agent.
Also contemplated is a cytotoxic conjugate, comprising one or more cytotoxic agents covalently bonded to a cell-binding agent through a PEG linking group having 41 to 1000 monomeric units, wherein a linkage of one of said one or more cytotoxic agents is illustrated in formula 3: 
wherein Z is said cytotoxic agent;
wherein Q is R2COOxe2x80x94, R2R3NCOOxe2x80x94, R2OCOOxe2x80x94, R2Oxe2x80x94, R2CONR3xe2x80x94, R2R3Nxe2x80x94, R2OCONR3xe2x80x94, or Sxe2x80x94,
xe2x80x83wherein:
R2 is SCR4R5R6xe2x80x94,
R4, R5 and R6 are each H, linear alkyl, cyclic alkyl or branched alkyl, and may be the same or different,
R3 is H or a linear alkyl, cyclic alkyl or branched alkyl,
wherein n is an integer of from 41 to 1000;
wherein x is 1 or 2; and
wherein A is said cell-binding agent.
Also contemplated is the cytotoxic conjugate according to any one of the relevant examples above, wherein said cytotoxic agent is selected from the group consisting of an thiol-containing maytansinoid, thiol-containing taxane, thiol-containing CC-1065 analogue, thiol-containing daunorubicin analogue and thiol-containing doxorubicin analogue, and thiol-containing analogues or derivatives thereof, and said cell-binding agent is selected from the group consisting of a polyclonal antibody, monoclonal antibody, antibody fragment, interferon, lymphokine, hormone, growth factor, vitamin and nutrient-transport molecule.
Also contemplated is the cytotoxic conjugate according to the relevant examples above, wherein said thiol-containing maytansinoid is a C-3 thiol-containing maytansinoid.
Also contemplated is the cytotoxic conjugate according to the relevant examples above, wherein said C-3 thiol-containing maytansinoid is an N-methyl-alanine-containing C-3 thiol-containing maytansinoid.
Also contemplated is the cytotoxic conjugate according to the relevant examples above, wherein said N-methyl-alanine-containing C-3 thiol-containing maytansinoid is a compound selected from the following formula M1: 
wherein:
l is an integer of from 1 to 10; and
may is a maytansinoid.
Also contemplated is the cytotoxic conjugate according to the relevant examples above, wherein said N-methyl-alanine-containing C-3 thiol-containing maytansinoid is a compound selected from the following formula M2: 
wherein:
R1 and R2 are H, CH3 or CH2CH3, and may be the same or different;
m is 0, 1, 2 or 3; and
may is a maytansinoid.
Also contemplated is the cytotoxic conjugate according to the relevant examples above, wherein said N-methyl-alanine-containing C-3 thiol-containing maytansinoid is a compound selected from the following formula M3: 
wherein:
n is an integer of from 3 to 8; and
may is a maytansinoid.
Also contemplated is the cytotoxic conjugate according to the relevant examples above, wherein said N-methyl-alanine-containing C-3 thiol-containing maytansinoid is a N-methyl-alanine-containing C-3 thiol-containing maytansinol.
Also contemplated is the cytotoxic conjugate according to the relevant examples above, wherein said N-methyl-alanine-containing C-3 thiol-containing maytansinol is a dechloro maytansinol.
Also contemplated is the cytotoxic conjugate according to the relevant examples above, wherein said N-methyl-alanine-containing C-3 thiol-containing maytansinol is a compound selected from the following formula M6: 
wherein:
l is 1, 2 or 3;
Y0 is Cl or H; and
X3 is H or CH3.
Also contemplated is the cytotoxic conjugate according to the relevant examples above, wherein said C-3 thiol-containing:maytansinoid is an N-methyl-cysteine-containing C-3 thiol-containing maytansinoid.
Also contemplated is the cytotoxic conjugate according to the relevant examples above, wherein said N-methyl-cysteine-containing C-3 thiol-containing maytansinoid is a compound selected from the following formula M4: 
wherein:
o is 1, 2 or 3;
p is an integer of 0 to 10; and
may is a maytansinoid.
Also contemplated is the cytotoxic conjugate according to the relevant examples above, wherein said N-methyl-cysteine-containing C-3 thiol-containing maytansinoid is a N-methyl-cysteine-containing C-3 thiol-containing maytansinol.
Also contemplated is the cytotoxic conjugate according to the relevant examples above, wherein said N-methyl-cysteine-containing C-3 thiol-containing maytansinol is a dechloro maytansinol.
Also contemplated is the cytotoxic conjugate according to the relevant examples above, wherein said N-methyl-cysteine-containing C-3 thiol-containing maytansinol is a compound selected from the following formula M5: 
wherein:
o is 1, 2 or 3;
q is an integer of from 0 to 10;
Y0 is Cl or H; and
X3 is H or CH3.
Also contemplated is the cytotoxic conjugate according to the relevant examples above, wherein said thiol-containing taxane is a compound selected from the following formula T1: 
wherein:
R1 is H, an electron withdrawing group, or an electron donating group, and R1xe2x80x2 and R1xe2x80x3 are the same or different and are H, an electron withdrawing group, or an electron donating group;
R2 is heterocyclic, a linear, branched, or cyclic ester or ether having from 1 to 10 carbon atoms or a carbamate of the formula xe2x80x94CONR10R11, wherein R10 and R11 are the same or different and are H, linear, branched or cyclic alkyl having 1 to 10 carbon atoms or aryl;
R3 is an aryl, or a linear, branched, or cyclic alkyl having from 1 to 10 carbon atoms;
R4 is xe2x80x94OC(CH3)3 or phenyl;
R5 is a thiol moiety; and
R6 is heterocyclic, H, a linear, branched, or cyclic ester or ether having from 1 to 10 carbon atoms or a carbamate of the formula xe2x80x94CONR10R11, wherein R10 and R11 are the same or different and are H, linear, branched or cyclic alkyl having 1 to 10 carbon atoms or aryl.
Also contemplated is the compound of the relevant examples above, wherein R1 is F, NO2, CN, Cl, CHF2, CF3, xe2x80x94OCH3, xe2x80x94OCH2CH3, or NR7R8, wherein:
R7 and R8 are the same or different and are linear, branched, or cyclic alkyl having 1 to 10 carbon atoms or simple or substituted aryl having 1 to 10 carbon atoms.
Also contemplated is the compound of the relevant examples above, wherein R7 and R8 each has 1 to 4 carbon atoms.
Also contemplated is the compound of the relevant examples above, wherein R7 and R8 are the same.
Also contemplated is the compound of the relevant examples above, wherein R7 and R8 are the same.
Also contemplated is the compound of the relevant examples above, wherein R2 is xe2x80x94COC2H5, xe2x80x94CH2CH3, xe2x80x94CONHCH2CH3xe2x80x94CO-morpholino, xe2x80x94CO-piperidino, xe2x80x94CO-piperazino, or xe2x80x94CO-N-methylpiperazino.
Also contemplated is the compound of the relevant examples above, wherein R5 is xe2x80x94(CH2)nS, xe2x80x94CO(CH2)nS, xe2x80x94(CH2)nCH(CH3)S, xe2x80x94CO(CH2)nCH(CH3)S, xe2x80x94(CH2)nC(CH3)2S, xe2x80x94CO(CH2)nC(CH3)2S, xe2x80x94CONR12(CH2)nS, xe2x80x94CONR12(CH2)nCH(CH3)S, xe2x80x94CONR12(CH2)nC(CH3)2S, xe2x80x94CO-morpholino-XS, xe2x80x94CO-piperidino-XS, xe2x80x94CO-piperazino-XS, or xe2x80x94CO-N-methylpiperazino-XS;
wherein n is an integer of 1 to 10; and
wherein R12 is H, a linear alkyl, branched alkyl or cyclic alkyl having 1 to 10 carbon atoms, or simple or substituted aryl having from 1 to 10 carbon atoms or heterocyclic.
Also contemplated is the compound of the relevant examples above, wherein R1 is in the meta position when R1xe2x80x2 and R1xe2x80x3 are H or OCH3.
Also contemplated is the compound of the relevant examples above, wherein R3 is xe2x80x94CHxe2x95x90C(CH3)2.
Also contemplated is the cytotoxic conjugate according to the relevant examples above, wherein said thiol-containing taxane is a compound selected from the following formula T1: 
wherein:
R1 is H, an electron withdrawing group, or an electron donating group, and R1xe2x80x2 and R1xe2x80x3 are the same or different and are H, an electron withdrawing group, or an electron donating group;
R2 is a thiol moiety;
R3 is aryl, or is a linear, branched, or cyclic alkyl having from 1 to 10 carbon atoms;
R4 is xe2x80x94OC(CH3)3 or phenyl;
R5 is heterocyclic, H, a linear, branched, or cyclic ester or ether having from 1 to 10 carbon atoms or a carbamate of the formula xe2x80x94CONR10R11, wherein R10 and R11 are the same or different and are H, linear, branched or cyclic alkyl having 1 to 10 carbon atoms or aryl; and
R6 is heterocyclic, H, a linear, branched, or cyclic ester or ether having from 1 to 10 carbon atoms or a carbamate of the formula xe2x80x94CONR10R11, wherein R10 and R11 are the same or different and are H, linear, branched or cyclic alkyl having 1 to 10 carbon atoms or aryl.
Also contemplated is the compound of the relevant examples above, wherein at least one of R1 is F, NO2, CN, Cl, CHF2, CF3, xe2x80x94OCH3, xe2x80x94OCH2CH3, or NR7R8 wherein:
R7 and R8 are the same or different and are linear, branched, or cyclic alkyl having 1 to 10 carbon atoms or simple or substituted aryl having 1 to 10 carbon atoms.
Also contemplated is the compound of the relevant examples above, wherein R7 and R8 each has 1 to 4 carbon atoms.
Also contemplated is the compound of the relevant examples above, wherein R7 and R8 are the same.
Also contemplated is the compound of the relevant examples above, wherein R7 and R8 are the same.
Also contemplated is the compound of the relevant examples above, wherein R5 is xe2x80x94(CH2)nS, xe2x80x94CO(CH2)nS, xe2x80x94(CH2)nCH(CH3)S, xe2x80x94CO(CH2)nCH(CH3)S, xe2x80x94(CH2)nC(CH3)2S, xe2x80x94CO(CH2)nC(CH3)2S, xe2x80x94CONR12(CH2)nS, xe2x80x94CONR12(CH2)nCH(CH3)S, xe2x80x94CONR12(CH2)nC(CH3)2S, xe2x80x94CO-morpholino-XS, xe2x80x94CO-piperidino-XS, xe2x80x94CO-piperazino-XS, or xe2x80x94CO-N-methylpiperazino-XS;
wherein n is an integer of 1 to 10; and
wherein R12 is H, a linear alkyl, branched alkyl or cyclic alkyl having 1 to 10 carbon atoms, or simple or substituted aryl having from 1 to 10 carbon atoms or heterocyclic.
Also contemplated is the compound of the relevant examples above, wherein R5 is xe2x80x94COC2H5, xe2x80x94CH2CH3, xe2x80x94CONHCH2CH3, xe2x80x94CO-morpholino, xe2x80x94CO-piperidino, xe2x80x94CO-piperazino, or xe2x80x94CO-N-methylpiperazino.
Also contemplated is the compound of the relevant examples above, wherein R1 is in the meta position when R1xe2x80x2 and R1xe2x80x3 are H or OCH3.
Also contemplated is the cytotoxic conjugate according to the relevant examples above, wherein said thiol-containing taxane is a compound selected from the following formula T1: 
wherein:
R1 is H, an electron withdrawing group, or an electron donating group, and R1xe2x80x2 and R1xe2x80x3 are the same or different and are H, an electron withdrawing group, or an electron donating group;
R2 is heterocyclic, a linear, branched, or cyclic ester or ether having from 1 to 10 carbon atoms or a carbamate of the formula xe2x80x94CONR10R11, wherein R10 and R11 are the same or different and are H, linear, branched or cyclic alkyl having 1 to 10 carbon atoms or aryl;
R3 is aryl, or is a linear, branched or cyclic alkyl having from 1 to 10 carbon atoms;
R4 is xe2x80x94OC(CH3)3 or phenyl;
R5 is heterocyclic, H, a linear, branched, or cyclic ester or ether having from 1 to 10 carbon atoms or a carbamate of the formula xe2x80x94CONR10R11, wherein R10 and R11 are the same or different and are H, linear, branched or cyclic alkyl having 1 to 10 carbon atoms or aryl;
R6 is a thiol moiety.
Also contemplated is the compound of the relevant examples above, wherein R1 is F, NO2, CN, Cl, CHF2, CF3, xe2x80x94OCH3, xe2x80x94OCH2CH3, or NR7R8 wherein:
R7 and R8 are the same or different and are linear, branched, or cyclic alkyl having 1 to 10 carbon atoms or simple or substituted aryl having 1 to 10 carbon atoms.
Also contemplated is the compound of the relevant examples above, wherein R7 and R8 each has 1 to 4 carbon atoms.
Also contemplated is the compound of the relevant examples above, wherein R7 and R8 are the same.
Also contemplated is the compound of the relevant examples above, wherein R7 and R8 are the same.
Also contemplated is the compound of the relevant examples above, wherein R2 is xe2x80x94COC2H5, xe2x80x94CH2CH3, xe2x80x94CONHCH2CH3, xe2x80x94CO-morpholino, xe2x80x94CO-piperidino, xe2x80x94CO-piperazino, or xe2x80x94CO-N-methylpiperazino.
Also contemplated is the compound of the relevant examples above, wherein R5 is xe2x80x94(CH2)nS, xe2x80x94CO(CH2)nS, xe2x80x94(CH2)nCH(CH3)S, xe2x80x94CO(CH2)nCH(CH3)S, xe2x80x94(CH2)nC(CH3)2S, xe2x80x94CO(CH2)nC(CH3)2S, xe2x80x94CONR12(CH2)nS, xe2x80x94CONR12(CH2)nCH(CH3)S, xe2x80x94CONR12(CH2)nC(CH3)2S, xe2x80x94CO-morpholino-XS, xe2x80x94CO-piperidino-XS, xe2x80x94CO-piperazino-XS, or xe2x80x94CO-N-methylpiperazino-XS;
wherein n is an integer of 1 to 10; and
wherein R12 is H, a linear alkyl, branched alkyl or cyclic alkyl having 1 to 10 carbon atoms, or simple or substituted aryl having from 1 to 10 carbon atoms or heterocyclic.
Also contemplated is the compound of the relevant examples above, wherein R1 is in the meta position when R1xe2x80x2 and R1xe2x80x3 are H or OCH3.
Also contemplated is the cytotoxic conjugate according to the relevant examples above, wherein said thiol-containing CC-1065 analogue is a cyclopropylbenzindole-containing cytotoxic compound formed from an A subunit of the formulae A-3 or A-4 covalently linked to either a B subunit of the formula F-1 or a B-C subunit of the formulae F-3 or F-7 via an amide bond from the secondary amino group of the pyrrole moiety of the A subunit to the C-2 carboxyl group of the B subunit,
wherein the formulae A-3 and A-4 are as follows: 
wherein the formulae F-1, F-3 and F-7 are as follows: 
wherein each Z may be the same or different and may be O or NH; and
wherein, in Formula F-1 R4 is a thiol moiety, in Formula F-3 one of R or R4 is a thiol moiety, in Formula F-7 one of Rxe2x80x2 or R4 is a thiol moiety; when R or Rxe2x80x2 is a thiol moiety, then R1 to R6, which may be the same or different, are hydrogen, C1-C3 linear alkyl, methoxy, hydroxyl, primary amino, secondary amino, tertiary amino, or amido; and when R4 is a thiol moiety, R, R1, R2, R3, R4, R5 and R6, which may be the same or different, are hydrogen, C1-C3 linear alkyl, methoxy, hydroxyl, primary amino, secondary amino, tertiary amino, or amido, and Rxe2x80x2 is NH2, alkyl, O-alkyl, primary amino, secondary amino, tertiary amino, or amido.
Also contemplated is the cytotoxic conjugate of the relevant examples above, wherein R and Rxe2x80x2 are thiol moieties and R1 to R6 are each hydrogen.
Also contemplated is the cytotoxic conjugate of the relevant examples above, wherein R or R4 is xe2x80x94NHCO(CH2)lS, xe2x80x94NHCOC6H4(CH2)lS, or xe2x80x94O(CH2)lS, and Rxe2x80x2 is xe2x80x94(CH2)lS, xe2x80x94NH(CH2)lS or xe2x80x94O(CH2)lS wherein:
l is an integer of 1 to 10.
Also contemplated is the cytotoxic conjugate of the relevant examples above, wherein R or R4 is xe2x80x94NHCO(CH2)lS, xe2x80x94NHCOC6H4(CH2)lS, or xe2x80x94O(CH2)lS, and Rxe2x80x2 is xe2x80x94(CH2)lS, xe2x80x94NH(CH2)lS or xe2x80x94O(CH2)lS wherein:
l is an integer of 1 to 10.
Also contemplated is the cytotoxic conjugate of the relevant examples above, wherein l is 1.
Also contemplated is the cytotoxic conjugate of the relevant examples above, wherein l is 2.
Also contemplated is the cytotoxic conjugate according to the relevant examples above, wherein said thiol-containing doxorubicin analogue is a compound selected from the following formula D2: 
wherein,
Y is O or NR2, wherein R2 is linear or branched alkyl having 1 to 5 carbon atoms;
R is a thiol moiety, H, or liner or branched alkyl having 1 to 5 carbon atoms; and
Rxe2x80x2 is a thiol moiety, H, or xe2x80x94OR1, wherein R1 is linear or branched alkyl having 1 to 5 carbon atoms;
provided that R and Rxe2x80x2 are not thiol moieties at the same time.
Also contemplated is the compound of the relevant examples above, wherein NR2 is NCH3.
Also contemplated is the compound of the relevant examples above, wherein Rxe2x80x2 is xe2x80x94O.
Also contemplated is the compound of the relevant examples above, wherein the thiol moiety is xe2x80x94(CH2)nS, xe2x80x94O(CH2)nS, xe2x80x94(CH2)nCH(CH3)S, xe2x80x94O(CH2)nCH(CH3)S, xe2x80x94(CH2)nC(CH3)2S, or xe2x80x94O(CH2)nC(CH3)2S, wherein n is an integer of 1 to 10.
Also contemplated is the cytotoxic conjugate according to the relevant examples above, wherein said thiol-containing daunorubicin analogue is a compound selected from the following 
wherein,
Y is O or NR2, wherein R2 is linear or branched alkyl having 1 to 5 carbon atoms;
R is a thiol moiety, H, or liner or branched alkyl having 1 to 5 carbon atoms; and
Rxe2x80x2 is a thiol moiety, H, or xe2x80x94OR1, wherein R1 is linear or branched alkyl having 1 to 5 carbon atoms;
provided that R and Rxe2x80x2 are not thiol moieties at the same time.
Also contemplated is the compound of the relevant examples above, wherein NR2 is NCH3.
Also contemplated is the compound of the relevant examples above, wherein Rxe2x80x2 is xe2x80x94O.
Also contemplated is the compound of the relevant examples above, wherein the thiol moiety is xe2x80x94(CH2)nS, xe2x80x94O(CH2)nS, xe2x80x94(CH2)nCH(CH3)S, xe2x80x94O(CH2)nCH(CH3)S, xe2x80x94(CH2)nC(CH3)2S, or xe2x80x94O(CH2)nC(CH3)2S, wherein n is an integer of 1 to 10.
Also contemplated is the cytotoxic conjugate according to the relevant examples above, wherein said cell-binding agent is a monoclonal antibody.
Also contemplated is the cytotoxic conjugate according to the relevant examples above, wherein said cell-binding agent is an antibody fragment.
Also contemplated is the cytotoxic conjugate according to the relevant examples above, wherein said cytotoxic agent is a taxane and said cell-binding agent is a monoclonal antibody.
In a third embodiment of the invention, a therapeutic composition comprising a therapeutically-effective amount of one of the cytotoxic conjugates of the present invention, and a pharmaceutically-acceptable carrier, is disclosed.
Specifically contemplated is a therapeutic composition comprising a therapeutically-effective amount of the cytotoxic conjugate of the relevant examples above, and a pharmaceutically acceptable carrier.
Also contemplated is the therapeutic composition according to the relevant examples above, wherein the therapeutically-effective amount of the cytotoxic conjugate is from 10 ug to 100 mg.
Also contemplated is the therapeutic composition according to the relevant examples above, wherein the therapeutically-effective amount of the cytotoxic conjugate is from 50 ug to 30 mg.
Also contemplated is the therapeutic composition according to the relevant examples above, wherein the therapeutically-effective amount of the cytotoxic conjugate is from 1 mg to 20 mg.
In a fourth embodiment of the invention, a method for producing a cytotoxic agent bearing a PEG linking group having a terminal active ester is disclosed. The method comprises a) reacting a PEG linking group through a disulfide group with a thiol-containing cytotoxic agent, and b) converting the terminal carboxylic acid group or protective chemical group of the product of step a) to an active ester, thereby producing a cytotoxic agent bearing a PEG linking group having a terminal active ester.
In a preferred embodiment, the PEG linking group has from 1 to 20 monomeric units. In an equally preferred embodiment, the PEG linking group has from 21 to 40 monomeric units. In a further equally preferred embodiment, the PEG linking group has from 41 to 1000 monomeric units.
Specifically contemplated is a method for producing a cytotoxic agent, bearing a PEG linking group having a terminal active ester and 1 to 20 monomeric units, of formula 2: 
wherein Z is said cytotoxic agent;
wherein Q is R2COOxe2x80x94, R2R3NCOOxe2x80x94, R2OCOOxe2x80x94, R2Oxe2x80x94, R2CONR3xe2x80x94, R2R3Nxe2x80x94, R2OCONR3xe2x80x94, or Sxe2x80x94,
xe2x80x83wherein:
R2 is SCR4R5R6xe2x80x94,
R4, R5 and R6 are each H, linear alkyl, cyclic alkyl or branched alkyl, and may be the same or different,
R3 is H or a linear alkyl, cyclic alkyl or branched alkyl;
wherein n is an integer of from 0 to 20;
wherein x is 1 or 2; and
wherein Y is N-succinimidyl, N-sulfosuccinimidyl, N-phthalimidyl, N-sulfophthalimidyl, 2-nitrophenyl, 4-nitrophenyl, 2,4-dinitrophenyl, 3-sulfonyl-4-nitrophenyl or 3-carboxy-4-nitrophenyl,
said method comprising the steps of:
a) reacting a PEG linking group having 1 to 20 monomeric units of formula 1: 
wherein Rxe2x80x2 is 2-pyridyl, 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro-4-pyridyl, 2-nitrophenyl, 4-nitrophenyl or 2,4-dinitrophenyl;
wherein Q is R2COOxe2x80x94, R2R3NCOOxe2x80x94, R2OCOOxe2x80x94, R2Oxe2x80x94, R2CONR3xe2x80x94, R2R3Nxe2x80x94, R2OCONR3xe2x80x94, or Sxe2x80x94,
xe2x80x83wherein:
R2 is SCR4R5R6xe2x80x94,
R4, R5 and R6 are each H, linear alkyl, cyclic alkyl or branched alkyl, and may be the same or different,
R3 is H or a linear alkyl, cyclic alkyl or branched alkyl;
wherein n is an integer of from 0 to 20;
wherein x is 1 or 2; and
wherein R is H, a cation to form a salt or a chemical group to form an ester, with a thiol-containing cytotoxic agent, and
b) converting the R group of the product of step a) to an active ester, thereby producing a cytotoxic agent bearing a PEG linking group having a terminal active ester and 1 to 20 monomeric units.
Also contemplated is a method for producing a cytotoxic agent, bearing a PEG linking group having a terminal active ester and 21 to 40 monomeric units, of formula 2: 
wherein Z is said cytotoxic agent;
wherein Q is R2COOxe2x80x94, R2R3NCOOxe2x80x94, R2OCOOxe2x80x94, R2Oxe2x80x94, R2CONR3xe2x80x94, R2R3Nxe2x80x94, R2OCONR3xe2x80x94, or Sxe2x80x94,
xe2x80x83wherein:
R2 is SCR4R5R6xe2x80x94,
R4, R5 and R6 are each H, linear alkyl, cyclic alkyl or branched alkyl, and may be the same or different,
R3 is H or a linear alkyl, cyclic alkyl or branched alkyl;
wherein n is an integer of from 21 to 40;
wherein x is 1 or 2; and
wherein Y is N-succinimidyl, N-sulfosuccinimidyl, N-phthalimidyl, N-sulfophthalimidyl, 2-nitrophenyl, 4-nitrophenyl, 2,4-dinitrophenyl, 3-sulfonyl-4-nitrophenyl or 3-carboxy-4-nitrophenyl,
said method comprising the steps of:
a) reacting a PEG linking group having 21 to 40 monomeric units of formula 1: 
wherein Rxe2x80x2 is 2-pyridyl, 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro-4-pyridyl, 2-nitrophenyl, 4-nitrophenyl or 2,4-dinitrophenyl;
wherein Q is R2COOxe2x80x94, R2R3NCOOxe2x80x94, R2OCOOxe2x80x94, R2Oxe2x80x94, R2CONR3xe2x80x94, R2R3Nxe2x80x94, R2OCONR3xe2x80x94, or Sxe2x80x94,
xe2x80x83wherein:
R2 is SCR4R5R6xe2x80x94,
R4, R5 and R6 are each H, linear alkyl, cyclic alkyl or branched alkyl, and may be the same or different,
R3 is H or a linear alkyl, cyclic alkyl or branched alkyl;
wherein n is an integer of from 21 to 40;
wherein x is 1 or 2; and
wherein R is H, a cation to form a salt or a chemical group to form an ester, with a thiol-containing cytotoxic agent, and
b) converting the R group of the product of step a) to an active ester, thereby producing a cytotoxic agent bearing a PEG linking group having a terminal active ester and 21 to 40 monomeric units.
Also contemplated is a method for producing a cytotoxic agent, bearing a PEG linking group having a terminal active ester and 41 to 1000 monomeric units, of formula 2: 
wherein Z is said cytotoxic agent;
wherein Q is R2COOxe2x80x94, R2R3NCOOxe2x80x94, R2OCOOxe2x80x94, R2Oxe2x80x94, R2CONR3xe2x80x94, R2R3Nxe2x80x94, R2OCONR3xe2x80x94, or Sxe2x80x94,
xe2x80x83wherein:
R2 is SCR4R5R6xe2x80x94,
R4, R5 and R6 are each H, linear alkyl, cyclic alkyl or branched alkyl, and may be the same or different,
R3 is H or a linear alkyl, cyclic alkyl or branched alkyl;
wherein n is an integer of from 41 to 1000;
wherein x is 1 or 2; and
wherein Y is N-succinimidyl, N-sulfosuccinimidyl, N-phthalimidyl, N-sulfophthalimidyl, 2-nitrophenyl, 4-nitrophenyl, 2,4-dinitrophenyl, 3-sulfonyl-4-nitrophenyl or 3-carboxy-4-nitrophenyl,
said method comprising the steps of:
a) reacting a PEG linking group having 41 to 1000 monomeric units of formula 1: 
wherein Rxe2x80x2 is 2-pyridyl, 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro-4-pyridyl, 2-nitrophenyl, 4-nitrophenyl or 2,4-dinitrophenyl;
wherein Q is R2COOxe2x80x94, R2R3NCOOxe2x80x94, R2OCOOxe2x80x94, R2Oxe2x80x94, R2CONR3xe2x80x94, R2R3Nxe2x80x94, R2OCONR3xe2x80x94, or Sxe2x80x94,
xe2x80x83wherein:
R2 is SCR4R5R6xe2x80x94,
R4, R5 and R6 are each H, linear alkyl, cyclic alkyl or branched alkyl, and may be the same or different,
R3 is H or a linear alkyl, cyclic alkyl or branched alkyl;
wherein n is an integer of from 41 to 1000;
wherein x is 1 or 2; and
wherein R is H, a cation to form a salt or a chemical group to form an ester, with a thiol-containing cytotoxic agent, and
b) converting the R group of the product of step a) to an active ester, thereby producing a cytotoxic agent bearing a PEG linking group having a terminal active ester and 41 to 1000 monomeric units.
Also contemplated is the method for producing a cytotoxic agent, bearing a PEG linking group having a terminal active ester, according to anyone of the relevant examples above, wherein the chemical group is methyl, ethyl, phenyl, benzyl or tertbutyl.
Also contemplated is the method for producing a cytotoxic agent, bearing a PEG linking group having a terminal active ester, according to any one of the relevant examples above, wherein said cytotoxic agent is selected from the group consisting of an thiol-containing maytansinoid, thiol-containing taxane, thiol-containing CC-1065 analogue, thiol-containing daunorubicin analogue and thiol-containing doxorubicin analogue, and thiol-containing analogues or derivatives thereof.
Also contemplated is the method according to the relevant examples above, wherein said thiol-containing maytansinoid is a C-3 thiol-containing maytansinoid.
Also contemplated is the method according to the relevant examples above, wherein said C-3 thiol-containing maytansinoid is an N-methyl-alanine-containing C-3 thiol-containing maytansinoid.
Also contemplated is the method according to the relevant examples above, wherein said N-methyl-alanine-containing C-3 thiol-containing maytansinoid is a compound selected from the following formula M1: 
wherein:
l is an integer of from 1 to 10; and
may is a maytansinoid.
Also contemplated is the method according to the relevant examples above, wherein said N-methyl-alanine-containing C-3 thiol-containing maytansinoid is a compound selected from the following formula M2: 
wherein:
R1 and R2 are H, CH3 or CH2CH3, and may be the same or different;
m is 0, 1, 2 or 3; and
may is a maytansinoid.
Also contemplated is the method according to the relevant examples above, wherein said N-methyl-alanine-containing C-3 thiol-containing maytansinoid is a compound selected from the following formula M3: 
wherein:
n is an integer of from 3 to 8; and
may is a maytansinoid.
Also contemplated is the method according to the relevant examples above, wherein said N-methyl-alanine-containing C-3 thiol-containing maytansinoid is a N-methyl-alanine-containing C-3 thiol-containing maytansinol.
Also contemplated is the method according to the relevant examples above, wherein said N-methyl-alanine-containing C-3 thiol-containing maytansinol is a dechloro maytansinol.
Also contemplated is the method according to the relevant examples above, wherein said N-methyl-alanine-containing C-3 thiol-containing maytansinol is a compound selected from the following formula M6: 
wherein:
l is 1 , 2 or 3;
Y0 is Cl or H; and
X3 is H or CH3.
Also contemplated is the method according to the relevant examples above, wherein said C-3 thiol-containing maytansinoid is an N-methyl-cysteine-containing C-3 thiol-containing maytansinoid.
Also contemplated is the method according to the relevant examples above, wherein said N-methyl-cysteine-containing C-3 thiol-containing maytansinoid is a compound selected from the following formula M4: 
wherein:
o is 1, 2 or 3;
p is an integer of 0 to 10; and
may is a maytansinoid.
Also contemplated is the method according to the relevant examples above, wherein said N-methyl-cysteine-containing C-3 thiol-containing maytansinoid is a N-methyl-cysteine-containing C-3 thiol-containing maytansinol.
Also contemplated is the method according to the relevant examples above, wherein said N-methyl-cysteine-containing C-3 thiol-containing maytansinol is a dechloro maytansinol.
Also contemplated is the method according to the relevant examples above, wherein said N-methyl-cysteine-containing C-3 thiol-containing maytansinol is a compound selected from the following formula M5: 
wherein:
o is 1, 2 or 3;
q is an integer of from 0 to 10;
Y0 is Cl or H; and
X3 is H or CH3.
Also contemplated is the method according to the relevant examples above, wherein said thiol-containing taxane is a compound selected from the following formula T1: 
wherein:
R1 is H, an electron withdrawing group, or an electron donating group, and R1xe2x80x2 and R1xe2x80x3 are the same or different and are H, an electron withdrawing group, or an electron donating group;
R2 is heterocyclic, a linear, branched, or cyclic ester or ether having from 1 to 10 carbon atoms or a carbamate of the formula xe2x80x94CONR10R11, wherein R10 and R11 are the same or different and are H, linear, branched or cyclic alkyl having 1 to 10 carbon atoms or aryl;
R3 is an aryl, or a linear, branched, or cyclic alkyl having from 1 to 10 carbon atoms;
R4 is xe2x80x94OC(CH3)3 or phenyl;
R5 is a thiol moiety; and
R6 is heterocyclic, H, a linear, branched, or cyclic ester or ether having from 1 to 10 carbon atoms or a carbamate of the formula xe2x80x94CONR10R11, wherein R10 and R11 are the same or different and are H, linear, branched or cyclic alkyl having 1 to 10 carbon atoms or aryl.
Also contemplated is the method according to the relevant examples above, wherein R1 is F, NO2, CN, Cl, CHF2, CF3, xe2x80x94OCH3, xe2x80x94OCH2CH3, or NR7R8, wherein:
R7 and R8 are the same or different and are linear, branched, or cyclic alkyl having 1 to 10 carbon atoms or simple or substituted aryl having 1 to 10 carbon atoms.
Also contemplated is the method according to the relevant examples above, wherein R7 and R8 each has 1 to 4 carbon atoms.
Also contemplated is the method according to the relevant examples above, wherein R7 and R8 are the same.
Also contemplated is the method according to the relevant examples above, wherein R7 and R8 are the same.
Also contemplated is the method according to the relevant examples above, wherein R2 is xe2x80x94COC2H5, xe2x80x94CH2CH3, xe2x80x94CONHCH2CH3xe2x80x94CO-morpholino, xe2x80x94CO-piperidino, xe2x80x94CO-piperazino, or xe2x80x94CO-N-methylpiperazino.
Also contemplated is the method according to the relevant examples above, wherein R5 is xe2x80x94(CH2)nS, xe2x80x94CO(CH2)nS, xe2x80x94(CH2)nCH(CH3)S, xe2x80x94CO(CH2)nCH(CH3)S, xe2x80x94(CH2)nC(CH3)2S, xe2x80x94CO(CH2)nC(CH3)2S, xe2x80x94CONR12(CH2)nS, xe2x80x94CONR12(CH2)nCH(CH3)S, xe2x80x94CONR12(CH2)nC(CH3)2S, xe2x80x94CO-morpholino-XS, xe2x80x94CO-piperidino-XS, xe2x80x94CO-piperazino-XS, or xe2x80x94CO-N-methylpiperazino-XS;
wherein n is an integer of 1 to 10; and
wherein R12 is H, a linear alkyl, branched alkyl or cyclic alkyl having 1 to 10 carbon atoms, or simple or substituted aryl having from 1 to 10 carbon atoms or heterocyclic.
Also contemplated is the method according to the relevant examples above, wherein R1 is in the meta position when R1xe2x80x2 and R1xe2x80x3 are H or OCH3.
Also contemplated is the method according to the relevant examples above, wherein R3 is xe2x80x94CHxe2x95x90C(CH3)2.
Also contemplated is the method according to the relevant examples above, wherein said taxane is a compound selected from the following formula T1: 
wherein:
R1 is H, an electron withdrawing group, or an electron donating group, and R1xe2x80x2 and R1xe2x80x3 are the same or different and are H, an electron withdrawing group, or an electron donating group;
R2 is a thiol moiety;
R3 is an aryl, or is a linear, branched, or cyclic alkyl having from 1 to 10 carbon atoms;
R4 is xe2x80x94OC(CH3)3 or phenyl;
R5 is heterocyclic, H, a linear, branched, or cyclic ester or ether having from 1 to 10 carbon atoms or a carbamate of the formula xe2x80x94CONR10R11, wherein R10 and R11 are the same or different and are H, linear, branched or cyclic alkyl having 1 to 10 carbon atoms or aryl; and
R6 is heterocyclic, H, a linear, branched, or cyclic ester or ether having from 1 to 10 carbon atoms or a carbamate of the formula xe2x80x94CONR10R11, wherein R10 and R11 are the same or different and are H, linear, branched or cyclic alkyl having 1 to 10 carbon atoms or aryl.
Also contemplated is the method according to the relevant examples above, wherein at least one of R1 is F, NO2, CN, Cl, CHF2, CF3, xe2x80x94OCH3, xe2x80x94OCH2CH3, or NR7R8 wherein:
R7 and R8 are the same or different and are linear, branched, or cyclic alkyl having 1 to 10 carbon atoms or simple or substituted aryl having 1 to 10 carbon atoms.
Also contemplated is the method according to the relevant examples above, wherein R7 and R8 each has 1 to 4 carbon atoms.
Also contemplated is the method according to the relevant examples above, wherein R7 and R8 are the same.
Also contemplated is the method according to the relevant examples above, wherein R7 and R8 are the same.
Also contemplated is the method according to the relevant examples above, wherein R5 is xe2x80x94(CH2)nS, xe2x80x94CO(CH2)nS, xe2x80x94(CH2)nCH(CH3)S, xe2x80x94CO(CH2)nCH(CH3)S, xe2x80x94(CH2)nC(CH3)2S, xe2x80x94CO(CH2)nC(CH3)2S, xe2x80x94CONR12(CH2)nS, xe2x80x94CONR12(CH2)nCH(CH3)S, xe2x80x94CONR12(CH2)nC(CH3)2S, xe2x80x94CO-morpholino-XS, xe2x80x94CO-piperidino-XS, xe2x80x94CO-piperazino-XS, or xe2x80x94CO-N-methylpiperazino-XS;
wherein n is an integer of 1 to 10; and
wherein R12 is H, a linear alkyl, branched alkyl or cyclic alkyl having 1 to 10 carbon atoms, or simple or substituted aryl having from 1 to 10 carbon atoms or heterocyclic.
Also contemplated is the method according to the relevant examples above, wherein R5 is xe2x80x94COC2H5, xe2x80x94CH2CH3, xe2x80x94CONHCH2CH3, xe2x80x94CO-morpholino, xe2x80x94CO-piperidino, xe2x80x94CO-piperazino, or xe2x80x94CO-N-methylpiperazino.
Also contemplated is the method according to the relevant examples above, wherein R1 is in the meta position when R1xe2x80x2 and R1xe2x80x3 are H or OCH3.
Also contemplated is the method according to the relevant examples above, wherein said taxane is a compound selected from the following formula T1: 
wherein:
R1 is H, an electron withdrawing group, or an electron donating group, and R1xe2x80x2 and R1xe2x80x3 are the same or different and are H, an electron withdrawing group, or an electron donating group;
R2 is heterocyclic, a linear, branched, or cyclic ester or ether having from 1 to 10 carbon atoms or a carbamate of the formula xe2x80x94CONR10R11, wherein R10 and R11 are the same or different and are H, linear, branched or cyclic alkyl having 1 to 10 carbon atoms or aryl;
R3 is an aryl, or is a linear, branched or cyclic alkyl having from 1 to 10 carbon atoms;
R4 is xe2x80x94OC(CH3)3 or phenyl;
R5 is heterocyclic, H, a linear, branched, or cyclic ester or ether having from 1 to 10 carbon atoms or a carbamate of the formula xe2x80x94CONR10R11, wherein R10 and R11 are the same or different and are H, linear, branched or cyclic alkyl having 1 to 10 carbon atoms or aryl;
R6 is a thiol moiety.
Also contemplated is the method according to the relevant examples above, wherein R1 is F, NO2, CN, Cl, CHF2, CF3, xe2x80x94OCH3, xe2x80x94OCH2CH3, or NR7R8 wherein:
R7 and R8 are the same or different and are linear, branched, or cyclic alkyl having 1 to 10 carbon atoms or simple or substituted aryl having 1 to 10 carbon atoms.
Also contemplated is the method according to the relevant examples above, wherein R7 and R8 each has 1 to 4 carbon atoms.
Also contemplated is the method according to the relevant examples above, wherein R7 and R8 are the same.
Also contemplated is the method according to the relevant examples above, wherein R7 and R8 are the same.
Also contemplated is the method according to the relevant examples above, wherein R2 is xe2x80x94COC2H5, xe2x80x94CH2CH3, xe2x80x94CONHCH2CH3, xe2x80x94CO-morpholino, xe2x80x94CO-piperidino, xe2x80x94CO-piperazino, or xe2x80x94CO-N-methylpiperazino.
Also contemplated is the method according to the relevant examples above, wherein R5 is xe2x80x94(CH2)nS, xe2x80x94CO(CH2)nS, xe2x80x94(CH2)nCH(CH3)S, xe2x80x94CO(CH2)nCH(CH3)S, xe2x80x94(CH2)nC(CH3)2S, xe2x80x94CO(CH2)nC(CH3)2S, xe2x80x94CONR12(CH2)nS, xe2x80x94CONR12(CH2)nCH(CH3)S, xe2x80x94CONR12(CH2)nC(CH3)2S, xe2x80x94CO-morpholino-XS, xe2x80x94CO-piperidino-XS, xe2x80x94CO-piperazino-XS, or xe2x80x94CO-N-methylpiperazino-XS;
wherein n is an integer of 1 to 10; and
wherein R12 is H, a linear alkyl, branched alkyl or cyclic alkyl having 1 to 10 carbon atoms, or simple or substituted aryl having from 1 to 10 carbon atoms or heterocyclic.
Also contemplated is the method according to the relevant examples above, wherein R1 is in the meta position when R1xe2x80x2 and R1xe2x80x3 are H or OCH3.
Also contemplated is the method according to the relevant examples above, wherein said thiol-containing CC-1065 analogue is acyclopropylbenzindole-containing cytotoxic compound formed from an A subunit of the formulae A-3 or A-4 covalently linked to either a B subunit of the formula F-1 or a B-C subunit of the formulae F-3 or F-7 via an amide bond from the secondary amino group of the pyrrole moiety of the A subunit to the C-2 carboxyl group of the B subunit,
wherein the formulae A-3 and A-4 are as follows: 
wherein the formulae F-1, F-3 and F-7 are as follows: 
wherein each Z may be the same or different and may be O or NH; and
wherein, in Formula F-1 R4 is a thiol moiety, in Formula F-3 one of R or R4 is a thiol moiety, in Formula F-7 one of Rxe2x80x2 or R4 is a thiol moiety; when R or Rxe2x80x2 is a thiol moiety, then R1 to R6, which may be the same or different, are hydrogen, C1-C3 linear alkyl, methoxy, hydroxyl, primary amino, secondary amino, tertiary amino, or amido; and when R4 is a thiol moiety, R, R1, R2, R3, R4, R5 and R6, which may be the same or different, are hydrogen, C1-C3 linear alkyl, methoxy, hydroxyl, primary amino, secondary amino, tertiary amino, or amido, and Rxe2x80x2 is NH2, alkyl, O-alkyl, primary amino, secondary amino, tertiary amino, or amido.
Also contemplated is the method according to the relevant examples above, wherein R and Rxe2x80x2 are thiol moieties and R1 to R6 are each hydrogen.
Also contemplated is the method according to the relevant examples above, wherein R or R4 is NHCO(CH2)lS, NHCOC6H4(CH2)nS, or O(CH2)lS, and Rxe2x80x2 is (CH2)lS, NH(CH2)lS or O(CH2)lS wherein:
l is an integer of 1 to 10.
Also contemplated is the method according to the relevant examples above, wherein R or R4 is NHCO(CH2)lS, NHCOC6H4(CH2)lS, or O(CH2)lS, and Rxe2x80x2 is (CH2)lS, NH(CH2)lS or O(CH2)lS wherein:
l is an integer of 1 to 10.
Also contemplated is the method according to the relevant examples above, wherein l is 1.
Also contemplated is the method according to the relevant examples above, wherein l is 2.
Also contemplated is the method according to the relevant examples above, wherein said thiol-containing doxorubicin analogue is a compound selected from the following formula D2: 
wherein,
Y is O or NR2, wherein R2 is linear or branched alkyl having 1 to 5 carbon atoms;
R is a thiol moiety, H, or liner or branched alkyl having 1 to 5 carbon atoms; and
Rxe2x80x2 is a thiol moiety, H, or xe2x80x94OR1, wherein R1 is linear or branched alkyl having 1 to 5 carbon atoms;
provided that R and Rxe2x80x2 are not thiol moieties at the same time.
Also contemplated is the method according to the relevant examples above, wherein NR2 is NCH3.
Also contemplated is the method according to the relevant examples above, wherein Rxe2x80x2 is xe2x80x94O.
Also contemplated is the method according to the relevant examples above, wherein the thiol moiety is xe2x80x94(CH2)nS, xe2x80x94O(CH2)nS, xe2x80x94(CH2)nCH(CH3)S, xe2x80x94O(CH2)nCH(CH3)S, xe2x80x94(CH2)nC(CH3)2S, or xe2x80x94O(CH2)nC(CH3)2S, wherein n is an integer of 1 to 10.
Also contemplated is the method according to the relevant examples above, wherein said thiol-containing daunorubicin analogue is a compound selected from the following formula D3: 
wherein,
Y is O or NR2, wherein R2 is linear or branched alkyl having 1 to 5 carbon atoms;
R is a thiol moiety, H, or liner or branched alkyl having 1 to 5 carbon atoms; and
Rxe2x80x2 is a thiol moiety, H, or xe2x80x94OR1, wherein R1 is linear or branched alkyl having 1 to 5 carbon atoms;
provided that R and Rxe2x80x2 are not thiol moieties at the same time.
Also contemplated is the method according to the relevant examples above, wherein NR2 is NCH3.
Also contemplated is the method according to the relevant examples above, wherein Rxe2x80x2 is xe2x80x94O.
Also contemplated is the method according to the relevant examples above, wherein the thiol moiety is xe2x80x94(CH2)nS, xe2x80x94O(CH2)nS, xe2x80x94(CH2)nCH(CH3)S, xe2x80x94O(CH2)nCH(CH3)S, xe2x80x94(CH2)nC(CH3)2S, or xe2x80x94O(CH2)nC(CH3)2S, wherein n is an integer of 1 to 10.
In a fifth embodiment of the invention, a method for producing a cytotoxic conjugate comprising one or more cytotoxic agents covalently bonded to a cell-binding agent through a PEG linking group is disclosed. The method comprises reacting one or more cytotoxic agents with a cell-binding agent, wherein each cytotoxic agent bears a PEG linking group having a terminal active ester, thereby producing a cytotoxic conjugate.
In a preferred embodiment, the PEG linking group has from 1 to 20 monomeric units. In an equally preferred embodiment, the PEG linking group has from 21 to 40 monomeric units. In a further equally preferred embodiment, the PEG linking group has from 41 to 1000 monomeric units.
Specifically contemplated is a method for producing a cytotoxic conjugate which comprises one or more cytotoxic agents covalently bonded to a cell-binding agent through a PEG linking group having 1 to 20 monomeric units, said method comprising reacting one or more cytotoxic agents with a cell-binding agent, wherein said one or more cytotoxic agents each bears a PEG linking group having a terminal active ester and 1 to 20 monomeric units, and wherein said cytotoxic agent bearing a PEG linking group having a terminal active ester and 1 to 20 monomeric units is a compound of formula 2: 
wherein Z is said cytotoxic agent;
wherein Q is R2COOxe2x80x94, R2R3NCOOxe2x80x94, R2OCOOxe2x80x94, R2Oxe2x80x94, R2CONR3xe2x80x94, R2R3Nxe2x80x94, R2OCONR3xe2x80x94, or Sxe2x80x94,
xe2x80x83wherein:
R2 is SCR4R5R6xe2x80x94,
R4, R5 and R6 are each H, linear alkyl, cyclic alkyl or branched alkyl, and may be the same or different,
R3 is H or a linear alkyl, cyclic alkyl or branched alkyl;
wherein n is an integer of from 0 to 20;
wherein x is 1 or 2; and
wherein Y is N-succinimidyl, N-sulfosuccinimidyl, N-phthalimidyl, N-sulfophthalimidyl, 2-nitrophenyl, 4-nitrophenyl, 2,4-dinitrophenyl, 3-sulfonyl-4-nitrophenyl or 3-carboxy-4-nitrophenyl,
thereby producing a cytotoxic conjugate, illustrated in formula 3 with a linkage of one of said one or more cytotoxic agents 
wherein Z is said cytotoxic agent;
wherein Q is R2COOxe2x80x94, R2R3NCOOxe2x80x94, R2OCOOxe2x80x94, R2Oxe2x80x94, R2CONR3xe2x80x94, R2R3Nxe2x80x94, R2OCONR3xe2x80x94, or Sxe2x80x94,
xe2x80x83wherein:
R2 is SCR4R5R6xe2x80x94,
R4, R5 and R6 are each H, linear alkyl, cyclic alkyl or branched alkyl, and may be the same or different,
R3 is H or a linear alkyl, cyclic alkyl or branched alkyl;
wherein n is an integer of from 0 to 20;
wherein x is 1 or 2; and wherein A is said cell-binding agent.
Also specifically contemplated is a method for producing a cytotoxic conjugate which comprises one or more cytotoxic agents covalently bonded to a cell-binding agent through a PEG linking group having 21 to 40 monomeric units, said method comprising reacting one or more cytotoxic agents with a cell-binding agent, wherein said one or more cytotoxic agents each bears a PEG linking group having a terminal active ester and 21 to 40 monomeric units, and wherein said cytotoxic agent bearing a PEG linking group having a terminal active ester and 21 to 40 monomeric units is a compound of formula 2: 
wherein Z is said cytotoxic agent;
wherein Q is R2COOxe2x80x94, R2R3NCOOxe2x80x94, R2OCOOxe2x80x94, R2Oxe2x80x94, R2CONR3xe2x80x94, R2R3Nxe2x80x94, R2OCONR3xe2x80x94, or Sxe2x80x94,
xe2x80x83wherein:
R2 is SCR4R5R6xe2x80x94,
R4, R5 and R6 are each H, linear alkyl, cyclic alkyl or branched alkyl, and may be the same or different,
R3 is H or a linear alkyl, cyclic alkyl or branched alkyl;
wherein n is an integer of from 21 to 40;
wherein x is 1 or 2; and
wherein Y is N-succinimidyl, N-sulfosuccinimidyl, N-phthalimidyl, N-sulfophthalimidyl, 2-nitrophenyl, 4-nitrophenyl, 2,4-dinitrophenyl, 3-sulfonyl-4-nitrophenyl or 3-carboxy-4-nitrophenyl,
thereby producing a cytotoxic conjugate, illustrated in formula 3 with a linkage of one of said one or more cytotoxic agents 
wherein Z is said cytotoxic agent;
wherein Q is R2COOxe2x80x94, R2R3NCOOxe2x80x94, R2OCOOxe2x80x94, R2Oxe2x80x94, R2CONR3xe2x80x94, R2R3Nxe2x80x94, R2OCONR3xe2x80x94, or Sxe2x80x94,
xe2x80x83wherein:
R2 is SCR4R5R6xe2x80x94,
R4, R5 and R are each H, linear alkyl, cyclic, alkyl or branched alkyl, and may be the same or different,
R3 is H or a linear alkyl, cyclic alkyl or branched alkyl;
wherein n is an integer of from 21 to 40;
wherein x is 1 or 2; and
wherein A is said cell-binding agent.
Also specifically contemplated is a method for producing a cytotoxic conjugate which comprises one or more cytotoxic agents covalently bonded to a cell-binding agent through a PEG linking group having 41 to 1000 monomeric units, said method comprising reacting one or more cytotoxic agents with a cell-binding agent, wherein said one or more cytotoxic agents each bears a PEG linking group having a terminal active ester and 41 to 1000 monomeric units, and wherein said cytotoxic agent bearing a PEG linking group having a terminal active ester and 41 to 1000 monomeric units is a compound of formula 2: 
wherein Z is said cytotoxic agent;
wherein Q is R2COOxe2x80x94, R2R3NCOOxe2x80x94, R2OCOOxe2x80x94, R2Oxe2x80x94, R2CONR3xe2x80x94, R2R3Nxe2x80x94, R2OCONR3xe2x80x94, or Sxe2x80x94,
xe2x80x83wherein:
R2 is SCR4R5R6xe2x80x94,
R4, R5 and R6 are each H, linear alkyl, cyclic alkyl or branched alkyl, and may be the same or different,
R3 is H or a linear alkyl, cyclic alkyl or branched alkyl;
wherein n is an integer of from 41 to 1000;
wherein x is 1 or 2; and
wherein Y is N-succinimidyl, N-sulfosuccinimidyl, N-phthalimidyl, N-sulfophthalimidyl, 2-nitrophenyl, 4-nitrophenyl, 2,4-dinitrophenyl, 3-sulfonyl-4-nitrophenyl or 3-carboxy-4-nitrophenyl,
thereby producing a cytotoxic conjugate, illustrated in formula 3 with a linkage of one of said one or more cytotoxic agents 
wherein Z is said cytotoxic agent;
wherein Q is R2COOxe2x80x94, R2R3NCOOxe2x80x94, R2OCOOxe2x80x94, R2Oxe2x80x94, R2CONR3xe2x80x94, R2R3Nxe2x80x94, R2OCONR3xe2x80x94, or Sxe2x80x94,
xe2x80x83wherein:
R2 is SCR4R5R6xe2x80x94,
R4, R5 and R6 are each H, linear alkyl, cyclic alkyl or branched alkyl, and may be the same or different,
R3 is H or a linear alkyl, cyclic alkyl or branched alkyl;
wherein n is an integer of from 41 to 1000;
wherein x is 1 or 2; and
wherein A is said cell-binding agent.
Also contemplated is the method for producing a cytotoxic conjugate according to any one of the relevant examples above, wherein said cytotoxic agent is selected from the group consisting of an thiol-containing maytansinoid, thiol-containing taxane, thiol-containing CC-1065 analogue, thiol-containing daunorubicin analogue and thiol-containing doxorubicin analogue, and thiol-containing analogues or derivatives thereof, and said cell-binding agent is selected from the group consisting of a polyclonal antibody, monoclonal antibody, antibody fragment, interferon, lymphokine, hormone, growth factor, vitamin and nutrient-transport molecule.
Also contemplated is the method according to the relevant examples above, wherein said thiol-containing maytansinoid is a C-3 thiol-containing maytansinoid.
Also contemplated is the method according to the relevant examples above, wherein said C-3 thiol-containing maytansinoid is an N-methyl-alanine-containing C-3 thiol-containing maytansinoid.
Also contemplated is the method according to the relevant examples above, wherein said N-methyl-alanine-containing C-3 thiol-containing maytansinoid is a compound selected from the following formula M1: 
wherein:
l is an integer of from 1 to 10; and
may is a maytansinoid.
Also contemplated is the method according to the relevant examples above, wherein said N-methyl-alanine-containing C-3 thiol-containing maytansinoid is a compound selected from the following formula M2: 
wherein:
R1 and R2 are H, CH3 or CH2CH3, and may be the same or different;
m is 0, 1, 2 or 3; and
may is a maytansinoid.
Also contemplated is the method according to the relevant examples above, wherein said N-methyl-alanine-containing C-3 thiol-containing maytansinoid is a compound selected from the following formula M3: 
wherein:
n is an integer of from 3 to 8; and
may is a maytansinoid.
Also contemplated is the method according to the relevant examples above, wherein said N-methyl-alanine-containing C-3 thiol-containing maytansinoid is a N-methyl-alanine-containing C-3 thiol-containing maytansinol.
Also contemplated is the method according to the relevant examples above, wherein said N-methyl-alanine-containing C-3 thiol-containing maytansinol is a dechloro maytansinol.
Also contemplated is the method according to the relevant examples above, wherein said N-methyl-alanine-containing C-3 thiol-containing maytansinol is a compound selected from the following formula M6: 
wherein:
l is 1, 2 or 3;
Y0 is Cl or H; and
X3 is H or CH3.
Also contemplated is the method according to the relevant examples above, wherein said C-3 thiol-containing maytansinoid is an N-methyl-cysteine-containing C-3 thiol-containing maytansinoid.
Also contemplated is the method according to the relevant examples above, wherein said N-methyl-cysteine-containing C-3 thiol-containing maytansinoid is a compound selected from the following formula M4: 
wherein:
o is 1, 2 or 3;
p is an integer of 0 to 10; and
may is a maytansinoid.
Also contemplated is the method according to the relevant examples above, wherein said N-methyl-cysteine-containing C-3 thiol-containing maytansinoid is a N-methyl-cysteine-containing C-3 thiol-containing maytansinol.
Also contemplated is the method according to the relevant examples above, wherein said N-methyl-cysteine-containing C-3 thiol-containing maytansinol is a dechloro maytansinol.
Also contemplated is the method according to the relevant examples above, wherein said N-methyl-cysteine-containing C-3 thiol-containing maytansinol is a compound selected from the following formula M5: 
wherein:
o is 1, 2 or 3;
q is an integer of from 0 to 10;
Y0 is Cl or H; and
X3 is H or CH3.
Also contemplated is the method according to the relevant examples above, wherein said thiol-containing taxane is a compound selected from the following formula T1: 
wherein:
R1 is H, an electron withdrawing group, or an electron donating group, and R1xe2x80x2 and R1xe2x80x3 are the same or different and are H, an electron withdrawing group, or an electron donating group;
R2 is heterocyclic, a linear, branched, or cyclic ester or ether having from 1 to 10 carbon atoms or a carbamate of the formula xe2x80x94CONR10R11, wherein R10 and R11 are the same or different and are H, linear, branched or cyclic alkyl having 1 to 10 carbon atoms or aryl;
R3 is an aryl, or a linear, branched, or cyclic alkyl having from 1 to 10 carbon atoms;
R4 is xe2x80x94OC(CH3)3 or phenyl;
R5 is a thiol moiety; and
R6 is heterocyclic, H, a linear, branched, or cyclic ester or ether having from 1 to 10 carbon atoms or a carbamate of the formula xe2x80x94CONR1OR11, wherein R10 and R11 are the same or different and are H, linear, branched or cyclic alkyl having 1 to 10 carbon atoms or aryl.
Also contemplated is the method according to the relevant examples above, wherein R1 is F, NO2, CN, Cl, CHF2, CF3, xe2x80x94OCH3, xe2x80x94OCH2CH3, or NR7R8, wherein:
R7 and R8 are the same or different and are linear, branched, or cyclic alkyl having 1 to 10 carbon atoms or simple or substituted aryl having 1 to 10 carbon atoms.
Also contemplated is the method according to the relevant examples above, wherein R7 and R8 each has 1 to 4 carbon atoms.
Also contemplated is the method according to the relevant examples above, wherein R7 and R8 are the same.
Also contemplated is the method according to the relevant examples above, wherein R7 and R8 are the same.
Also contemplated is the method according to the relevant examples above, wherein R2 is xe2x80x94COC2H5, xe2x80x94CH2CH3, xe2x80x94CONHCH2CH3xe2x80x94CO-morpholino, xe2x80x94CO-piperidino, xe2x80x94CO-piperazino, or xe2x80x94CO-N-methylpiperazino.
Also contemplated is the method according to the relevant examples above, wherein R5 is xe2x80x94(CH2)nS, xe2x80x94CO(CH2)nS, xe2x80x94(CH2)nCH(CH3)S, xe2x80x94CO(CH2)nCH(CH3)S, xe2x80x94(CH2)nC(CH3)2S, xe2x80x94CO(CH2)nC(CH3)2S, xe2x80x94CONR12(CH2)nS, xe2x80x94CONR12(CH2)nCH(CH3)S, xe2x80x94CONR12(CH2)nC(CH3)2S, xe2x80x94CO-morpholino-XS, xe2x80x94CO-piperidino-XS, xe2x80x94CO-piperazino-XS, or xe2x80x94CO-N-methylpiperazino-XS;
wherein n is an integer of 1 to 10; and
wherein R12 is H, a linear alkyl, branched alkyl or cyclic alkyl having 1 to 10 carbon atoms, or simple or substituted aryl having from 1 to 10 carbon atoms or heterocyclic.
Also contemplated is the method according to the relevant examples above, wherein R1 is in the meta position when R1xe2x80x2 and R1xe2x80x3 are H or OCH3.
Also contemplated is the method according to the relevant examples above, wherein R3 is xe2x80x94CHxe2x95x90C(CH3)2.
Also contemplated is the method according to the relevant examples above, wherein said taxane is a compound selected from the following formula T1: 
wherein:
R1 is H, an electron withdrawing group, or an electron donating group, and R1xe2x80x2 and R1xe2x80x3 are the same or different and are H, an electron withdrawing group, or an electron donating group;
R2 is a thiol moiety;
R3 is an aryl, or is a linear, branched, or cyclic alkyl having from 1 to 10 carbon atoms;
R4 is xe2x80x94OC(CH3)3 or phenyl;
R5 is heterocyclic, H, a linear, branched, or cyclic ester or ether having from 1 to 10 carbon atoms or a carbamate of the formula xe2x80x94CONR10R11, wherein R10 and R11 are the same or different and are H, linear, branched or cyclic alkyl having 1 to 10 carbon atoms or aryl; and
R6 is heterocyclic, H, a linear, branched, or cyclic ester or ether having from 1 to 10 carbon atoms or a carbamate of the formula xe2x80x94CONR10R11, wherein R10 and R11 are the same or different and are H, linear, branched or cyclic alkyl having 1 to 10 carbon atoms or aryl.
Also contemplated is the method according to the relevant examples above, wherein at least one of R1 is F, NO2, CN, Cl, CHF2, CF3, xe2x80x94OCH3, xe2x80x94OCH2CH3, or NR7R8 wherein:
R7 and R8 are the same or different and are linear, branched, or cyclic alkyl having 1 to 10 carbon atoms or simple or substituted aryl having 1 to 10 carbon atoms.
Also contemplated is the method according to the relevant examples above, wherein R7 and R8 each has 1 to 4 carbon atoms.
Also contemplated is the method according to the relevant examples above, wherein R7 and R8 are the same.
Also contemplated is the method according to the relevant examples above, wherein R7 and R8 are the same.
Also contemplated is the method according to the relevant examples above, wherein R5 is xe2x80x94(CH2)nS, xe2x80x94CO(CH2)nS, xe2x80x94(CH2)nCH(CH3)S, xe2x80x94CO(CH2)nCH(CH3)S, xe2x80x94(CH2)nC(CH3)2S, xe2x80x94CO(CH2)nC(CH3)2S, xe2x80x94CONR12(CH2)nS, xe2x80x94CONR12(CH2)nCH(CH3)S, xe2x80x94CONR12(CH2)nC(CH3)2S, xe2x80x94CO-morpholino-XS, xe2x80x94CO-piperidino-XS, xe2x80x94CO-piperazino-XS, or xe2x80x94CO-N-methylpiperazino-XS;
wherein n is an integer of 1 to 10; and
wherein R12 is H, a linear alkyl, branched alkyl or cyclic alkyl having 1 to 10 carbon atoms, or simple or substituted aryl having from 1 to 10 carbon atoms or heterocyclic.
Also contemplated is the method according to the relevant examples above, wherein R5 is xe2x80x94COC2H5, xe2x80x94CH2CH3, xe2x80x94CONHCH2CH3, xe2x80x94CO-morpholino, xe2x80x94CO-piperidino, xe2x80x94CO-piperazino, or xe2x80x94CO-N-methylpiperazino.
Also contemplated is the method according to the relevant examples above, wherein R1 is in the meta position when R1xe2x80x2 and R1xe2x80x3 are H or OCH3.
Also contemplated is the method according to the relevant examples above, wherein said taxane is a compound selected from the following formula T1: 
wherein:
R1 is H, an electron withdrawing group, or an electron donating group, and R1xe2x80x2 and R1xe2x80x3 are the same or different and are H, an electron withdrawing group, or an electron donating group;
R2 is heterocyclic, a linear, branched, or cyclic ester or ether having from 1 to 10 carbon atoms or a carbamate of the formula xe2x80x94CONR10R11, wherein R10 and R11 are the same or different and are H, linear, branched or cyclic alkyl having 1 to 10 carbon atoms or aryl;
R3 is an aryl, or is a linear, branched or cyclic alkyl having from 1 to 10 carbon atoms;
R4 is xe2x80x94OC(CH3)3 or phenyl;
R5 is heterocyclic, H, a linear, branched, or cyclic ester or ether having from 1 to 10 carbon atoms or a carbamate of the formula xe2x80x94CONR10R11, wherein R10 and R11 are the same or different and are H, linear, branched or cyclic alkyl having 1 to 10 carbon atoms or aryl;
R6 is a thiol moiety.
Also contemplated is the method according to the relevant examples above, wherein R1 is F, NO2, CN, Cl, CHF2, CF3, xe2x80x94OCH3, xe2x80x94OCH2CH3, or NR7R8 wherein:
R7 and R8 are the same or different and are linear, branched, or cyclic alkyl having 1 to carbon atoms or simple or substituted aryl having 1 to 10 carbon atoms.
Also contemplated is the method according to the relevant examples above, wherein R7 and R8 each has 1 to 4 carbon atoms.
Also contemplated is the method according to the relevant examples above, wherein R7 and R8 are the same.
Also contemplated is the method according to the relevant examples above, wherein R7 and R8 are the same.
Also contemplated is the method according to the relevant examples above, wherein R2 is xe2x80x94COC2H5, xe2x80x94CH2CH3, xe2x80x94CONHCH2CH3, xe2x80x94CO-morpholino, xe2x80x94CO-piperidino, xe2x80x94CO-piperazino, or xe2x80x94CO-N-methylpiperazino.
Also contemplated is the method according to the relevant examples above, wherein R5 is xe2x80x94(CH2)nS, xe2x80x94CO(CH2)nS, xe2x80x94(CH2)nCH(CH3)S, xe2x80x94CO(CH2)nCH(CH3)S, xe2x80x94(CH2)nC(CH3)2S, xe2x80x94CO(CH2)nC(CH3)2S, xe2x80x94CONR12(CH2)nS, xe2x80x94CONR12(CH2)nCH(CH3)S, xe2x80x94CONR12(CH2)nC(CH3)2S, xe2x80x94CO-morpholino-XS, xe2x80x94CO-piperidino-XS, xe2x80x94CO-piperazino-XS, or xe2x80x94CO-N-methylpiperazino-XS;
wherein n is an integer of 1 to 10; and
wherein R12 is H, a linear alkyl, branched alkyl or cyclic alkyl having 1 to 10 carbon atoms, or simple or substituted aryl having from 1 to 10 carbon atoms or heterocyclic.
Also contemplated is the method according to the relevant examples above, wherein R1 is in the meta position when R1xe2x80x2 and R1xe2x80x3 are H or OCH3.
Also contemplated is the method according to the relevant examples above, wherein said thiol-containing CC-1065 analogue is a cyclopropylbenzindole-containing cytotoxic compound formed from an A subunit of the formulae A-3 or A-4 covalently linked to either a B subunit of the formula F-1 or a B-C subunit of the formulae F-3 or F-7 via an amide bond from the secondary amino group of the pyrrole moiety of the A subunit to the C-2 carboxyl group of the B subunit,
wherein the formulae A-3 and A-4 are as follows: 
wherein the formulae F-1, F-3 and F-7 are as follows: 
wherein each Z may be the same or different and may be O or NH; and
wherein, in Formula F-1 R4 is a thiol moiety, in Formula F-3 one of R or R4 is a thiol moiety, in Formula F-7 one of Rxe2x80x2 or R4 is a thiol moiety; when R or Rxe2x80x2 is a thiol moiety, then R1 to R6, which may be the same or different, are hydrogen, C1-C3 linear alkyl, methoxy, hydroxyl, primary amino, secondary amino, tertiary amino, or amido; and when R4 is a thiol moiety, R, R1, R2, R3, R4, R5 and R6, which may be the same or different, are hydrogen, C1-C3 linear alkyl, methoxy, hydroxyl, primary amino, secondary amino, tertiary amino, or amido, and Rxe2x80x2 is NH2, alkyl, O-alkyl, primary amino, secondary amino, tertiary amino, or amido.
Also contemplated is the method according to the relevant examples above, wherein R and Rxe2x80x2 are thiol moieties and R1 to R6 are each hydrogen.
Also contemplated is the method according to the relevant examples above, wherein R or R4 is NHCO(CH2)lS, NHCOC6H4(CH2)lS, or O(CH2)lS, and Rxe2x80x2 is (CH2)lS, NH(CH2)lS or O(CH2)lS wherein:
l is an integer of 1 to 10.
Also contemplated is the method according to the relevant examples above, wherein R or R4 is NHCO(CH2)lS, NHCOC6H4(CH2)lS, or O(CH2)lS, and Rxe2x80x2 is (CH2)lS, NH(CH2)lS or O(CH2)lS wherein:
l is an integer of 1 to 10.
Also contemplated is the method according to the relevant examples above, wherein l is 1.
Also contemplated is the method according to the relevant examples above, wherein l is 2.
Also contemplated is the method according to the relevant examples above, wherein said thiol-containing doxorubicin analogue is a compound selected from the following formula D2: 
wherein,
Y is O or NR2, wherein R2 is linear or branched alkyl having 1 to 5 carbon atoms;
R is a thiol moiety, H, or liner or branched alkyl having 1 to 5 carbon atoms; and
Rxe2x80x2 is a thiol moiety, H, or xe2x80x94OR1, wherein R1 is linear or branched alkyl having 1 to 5 carbon atoms;
provided that R and Rxe2x80x2 are not thiol moieties at the same time.
Also contemplated is the method according to the relevant examples above, wherein NR2 is NCH3.
Also contemplated is the method according to the relevant examples above, wherein Rxe2x80x2 is xe2x80x94O.
Also contemplated is the method according to the relevant examples above, wherein the thiol moiety is xe2x80x94(CH2)nS, xe2x80x94O(CH2)nS, xe2x80x94(CH2)nCH(CH3)S, xe2x80x94O(CH2)nCH(CH3)S, xe2x80x94(CH2)nC(CH3)2S, or xe2x80x94O(CH2)nC(CH3)2S, wherein n is an integer of 1 to 10.
Also contemplated is the method according to the relevant examples above, wherein said thiol-containing daunorubicin analogue is a compound selected from the following formula D3: 
wherein,
Y is O or NR2, wherein R2 is linear or branched alkyl having 1 to 5 carbon atoms;
R is a thiol moiety, H, or liner or branched alkyl having 1 to 5 carbon atoms; and
Rxe2x80x2 is a thiol moiety, H, or OR1, wherein R1 is linear or branched alkyl having 1 to 5 carbon atoms;
provided that R and Rxe2x80x2 are not thiol moieties at the same time.
Also contemplated is the method according to the relevant examples above, wherein NR2 is NCH3.
Also contemplated is the method according to the relevant examples above, wherein Rxe2x80x2 is xe2x80x94O.
Also contemplated is the method according to the relevant examples above, wherein the thiol moiety is xe2x80x94(CH2)nS, xe2x80x94O(CH2)nS, xe2x80x94(CH2)nCH(CH3)S, xe2x80x94O(CH2)nCH(CH3)S, xe2x80x94(CH2)nC(CH3)2S, or xe2x80x94O(CH2)nC(CH3)2S, wherein n is an integer of 1 to 10.
Also contemplated is the method according to the relevant examples above, wherein said cell-binding agent is a monoclonal antibody.
Also contemplated is the method according to the relevant examples above, wherein said cell-binding agent is an antibody fragment.
Also contemplated is the method according to the relevant examples above, wherein said cytotoxic agent is a taxane and said cell-binding agent is a monoclonal antibody.
Also contemplated is the method according to any one of the relevant examples above, wherein said method further comprises the initial step of preparing a cytotoxic agent, bearing a PEG linking group having a terminal active ester, said initial step comprising reacting a thiol-containing cytotoxic agent with a PEG linking group of formula 1: 
wherein Rxe2x80x2 is 2-pyridyl, 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro-4-pyridyl, 2-nitrophenyl, 4-nitrophenyl or 2,4-dinitrophenyl;
wherein Q is R2COOxe2x80x94, R2R3NCOOxe2x80x94, R2OCOOxe2x80x94, R2Oxe2x80x94, R2CONR3xe2x80x94, R2R3Nxe2x80x94, R2OCONR3xe2x80x94, or Sxe2x80x94,
xe2x80x83wherein:
R2 is SCR4R5R6xe2x80x94,
R4, R5 and R6 are each H, linear alkyl, cyclic alkyl or branched alkyl, and may be the same or different,
R3 is H or a linear alkyl, cyclic alkyl or branched alkyl;
wherein n is an integer of from 0 to 20, n is an integer of from 21 to 40, or n is an integer of from 41 to 1000;
wherein x is 1 or 2; and
wherein R is H, a cation to form a salt or a chemical group to form an ester.
In a final embodiment of the invention, a method of killing selected cell populations comprising contacting target cells, or tissue containing target cells, with an effective amount of one of the cytotoxic conjugates or therapeutic compositions disclosed herein, is disclosed.
Specifically contemplated is a method of killing selected cell populations comprising contacting target cells, or tissue containing target cells, with an effective amount of the cytotoxic conjugate of the relevant examples above.